Cell-permeable reprogramming factor (iCP-RF) recombinant protein and use thereof

ABSTRACT

The iCP-RF recombinant protein of present invention could mediate generation of the induced pluripotent stem cells (iPSCs) from terminally differentiated somatic cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Bypass Continuation of International Application No. PCT/KR2016/008757 filed Aug. 9, 2016, claiming benefit of U.S. Provisional Patent Application No. 62/202,987 filed Aug. 10, 2015, the contents of all of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to providing improved cell-permeability reprogramming factor (iCP-RF) recombinant protein and uses thereof. The recombinant protein improved cell-permeability and biological activity as a generation of induced pluripotent stem cells (iPSCs) from terminally differentiated somatic cells.

BACKGROUND ART

Stem cells have been emerged as ideal cell sources in cell-based therapies because they possess great ability to differentiate into various lineages and self-renewability. Embryonic stem (ES) cells, which are established from the preimplantation embryos of mouse or human, can be cultures for extended periods while maintaining their pluripotent ability to differentiate into all kind of lineages of cells in the body. Human embryonic stem cells have the potential to be used to understand the mechanisms of disease, screen the efficacy and safety of novel drugs and treat various diseases, including leukemia and Parkinson's disease and to be used as regenerative cell therapy. However, in clinical trials, the embryonic stem cells transplantation caused severe rejection reactions equivalent to organ transplant rejection. Some are ethically opposed to the use of embryonic stem cells obtained by destroying human embryos. To avoid these ethical issues and cell availability, mesenchymal stem cells (MSCs) from adult tissues were suggested as alternatives for embryonic stem cells due to their multi-lineage differentiation potential and ability of unlimited self-renewal (1). MSCs can avoid immune rejection problem since they can be easily obtained from the patients' own various types of tissues, such as bone marrow, adipose tissue and periodontal ligaments. However, MSCs have shown limited differentiation potential into connective tissues including osteogenic, chondrogenic, and adipogenic lineages. Therefore, it was not sufficient to replace embryonic stem cells.

Yamanaka et al. reported that terminally differentiated somatic cells can be reprogrammed to the induced pluripotent stem cells (iPSCs), which possess pluripotency and self-renewability by enforced expression of reprogramming factors (2 and 3). Reprogramming factors (RFs) include transcription factors that require for the maintenance of embryonic stem cells in pluripotent status, OCT4 (Octamer-binding transcription factor 4), SOX2 (Sex determining region Y-box 2) and NANOG (Homeobox protein NANOG), as well as other proteins that facilitate self-renewal and inhibit differentiation of cells, CMYC (c-Myc), KLF4 (Kruppel-like factor 4) and LIN28 (Lin-28 homolog A) (4 and 5). Additionally, ZSCAN4 (Zinc finger and SCAN domain containing 4) plays important role in telomere elongation and genome stabilization which involves in immortalized cell line establishment (6 to 8). These reprogramming factors can be treated as sets: 1) “Yamanaka factor” including OCT4, SOX2, KLF4 and CMYC, and 2) “Thomson factor” including OCT4, SOX2, NANOG and LIN28 (3).

Patient-derived iPSCs are expected to be used for autologous stem cell therapy as an alternative of ES cells without any rejection reaction and the ethical issue of using ES cells. However, the efficiency of retro- or lenti-virus-mediated introduction of reprogramming factor genes into fibroblasts showed only ˜0.05% (9 and 10). In addition, it has a potential to cause mutation by the integration of vectors into the genome. Moreover, reprogramming factors that facilitate the formation of iPSCs have shown serious side effects, such as tumorigenesis by CMYC or epithelial dysplasia by enforced expression of OCT4 and KLF4. In terms of practicality, the application of iPSCs in the field of regenerative medicine requires more effective methods to avoid dysregulated RFs activity or vector-induced mutation that may occur during the introduction of reprogramming factors into the somatic cells.

These limitations have led to the development of various different methods to generate transgene free-iPSCs, including: (i) loxP flanked vectors (11), (ii) excisable transposons (12), (iii) adenovirus (13) and Sendai virus (14) vectors, and (iv) non-integrating episomal vectors (15). Adeno virus-mediated reprogramming factor integration shows 10-3 to 10-5 per cells of frequencies (16). Moreover, these methods have displayed problems such as incomplete deletion or continuous existing of exogenous genes. Although the reprogramming factor genes can be introduced into cells by using plasmid transfection, but it shows more than 100-fold lower efficiency than that of retrovirus transduction (9). Other approaches avoid DNA-based vectors to generate iPSCs, such as synthetic modified RNA (17), epigenetic regulation by chemical compounds (18) and direct uptake of RF proteins (19).

Therefore, introducing RF proteins could be considered as the only method to avoid the major obstacles with genetic damage and gene dysregulation caused by gene-based vectors and to provide more quantitatively and timely regulation of stem cell reprogramming. The initial protein-based RFs delivery-mediated by Tat protein transduction domain (PTD) that contains short basic arginine-rich region (aa 48-57) of HIV-1. Although the PTD fused-proteins can be transduced into the cells mediated by lipid raft-dependent micropinocytosis, most of Tat-fused proteins remain trapped in macropinosomes, caused by failure of proteins to escape from macropinosomes. Because of these limitations, Kim and Ding successfully reprogrammed mouse embryonic fibroblast (19) and human newborn fibroblast (20) cells to iPS cells by using poly-arginine (11R or 9R) PTD-fused reprogramming factors (OCT4, SOX2, KLF4, and CMYC), but they shows very low efficiency (0.001% to 0.006%).

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DISCLOSURE Technical Problem

A macromolecule, such as reprogramming factors (RFs), cannot be translocated across the cell membrane; furthermore, it cannot be transported into the nucleus of the cell. Therefore, there was a need to develop macromolecule intracellular transduction technology (MITT), which enables the translocation of macromolecules into the cell/tissues.

In the previous studies, MITT-based hydrophobic CPPs named membrane translocating sequence (MTS) and membrane translocating motif (MTM), derived from the hydrophobic signal peptide of fibroblast growth factor 4 (FGF4) have been reported and used to deliver biologically active peptides and proteins, such as reprogramming factors, systemically in animals.

However, they could not effectively deliver reprogramming factor (RF) protein in vitro were also insufficient due to protein aggregation, low solubility/yield and poor cell/tissue-permeability.

Technical Solution

To overcome the limitations and improve CPPs that provide cell-permeability of macromolecules in vitro and in vivo, theoretical critical factors (CFs) to improve the intracellular delivery potential of the CPPs are identified and verified according to one embodiment of the present invention. Based on the CFs determined, hydrophobic CPP sequences are newly created, quantitatively evaluated for cell-permeability and mutually compared to reference CPP sequences in their intracellular delivery potential in live cells. One embodiment of the present invention, newly developed hydrophobic CPPs are presented. The novel peptide sequences termed ‘advanced macromolecule transduction domains’ (aMTDs) could systematically deliver the aMTD-fused recombinant proteins to live cells and animal tissues.

One aspect of the present invention relates to baseline platform that could be applied to unlimited number of designs, having cell-permeability applicable for biomedical sciences, preclinical and clinical studies that facilitate the traverse of biologically active macromolecules, including proteins, peptides, nucleic acids, chemicals and so on, across the plasma membrane in cells.

The present inventors analyzed, identified, and determined these critical factors that facilitate the cell permeable ability of aMTD sequences. These aMTD sequences are artificially assembled based on the critical factors (CFs) determined from in-depth analysis of previously published hydrophobic CPPs.

One aspect of the present invention relates to novel advanced macromolecule transduction domain (aMTD) sequences.

The aMTD sequences of one aspect of the present invention are the first artificially developed cell permeable polypeptides capable of mediating the transduction of biologically active macromolecules—including peptides, polypeptides, protein domains, or full-length proteins—through the plasma membrane of cells.

Another aspect of the present invention relates to the method of genetically engineering a biologically active molecules having cell-permeability by fusing the aMTD sequences to the biologically active cargo molecules.

One aspect of the present invention also relates to its therapeutic application for the delivery of biologically active molecules to cells, involving cell-permeable recombinant proteins, where aMTDs are attached to the biologically active cargo molecules.

Another aspect of the present invention pertains to a method in which biologically active macromolecules are able to enter into live cells, as constructs of cell-permeable recombinant proteins comprised of aMTD sequences fused to biologically active macromolecules.

Other aspects of the present invention relate to an efficient use of aMTD sequences for molecule delivery, drug delivery, protein therapy, intracellular protein therapy, protein replacement therapy, peptide therapy, gene delivery and so on.

Another aspect of the present invention relates to 240 new hydrophobic CPP sequences—aMTDs, determination of the aMTD-mediated intracellular delivery activity of the recombinant proteins, and comparison of the enhanced protein uptake by live cells at levels greater than or equal to the FGF4-derived MTS/MTM and HRSS-derived MTD sequences. These strengths of newly invented aMTDs could address the setbacks on reference hydrophobic CPPs for clinical development and application.

One aspect of the present invention pertains to advanced macromolecule transduction domain (aMTD) sequences that transduce biologically active macromolecules into the plasma membrane.

Another aspect of the present invention directs to aMTD consisting of amino acid sequences having the following characteristics:

a. Amino acid length: 9 to 13

b. Bending potential: Proline (P) positioned in the middle (5′, 6′, 7′ or 8′) and at the end (12′) of the sequence.

c. Rigidity/Flexibility: Instability Index (II): 40 to 60

d. Structural Feature: Aliphatic Index (AI): 180 to 220

e. Hydropathy: GRAVY: 2.1 to 2.6

f. Amino acid composition: All of composed amino acids are hydrophobic and aliphatic amino acids (A, V, L, I and P) According to one embodiment, the amino acid sequences have the general formula composed of 12 amino acid sequences as described below. [General formula] X1-X2-X3-X4-U5-U6-U7-U8-X9-X10-X11-P

wherein (P) at the end of sequence (12′) is proline, one of U5, U6, U7, and U8 is proline, X(s) and U(s) which is not proline are A, V, L and/or I.

According to one embodiment, the amino acid sequences having the general formula are selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 240.

According to one embodiment, the secondary structure of the aMTD is α-Helix.

One aspect of the present invention further provides isolated polynucleotides that encode aMTD sequences described above.

According to one embodiment, the isolated polynucleotides are selected from the group consisting of SEQ ID NO: 241 to SEQ ID NO: 480.

Another aspect of the present invention further provides a method of identifying critical factors of aMTDs. The 6 methods comprise selecting superior hydrophobic CPPs from previously published reference hydrophobic CPPs; analyzing physiological and chemical characteristics of the selected hydrophobic CPPs; identifying features that are in association with cell-permeability out of these physiological and chemical characteristics; categorizing previously published reference hydrophobic CPPs into at least 2 groups and determining unique features by in-depth analysis of each groups of CPPs according to their cell-permeability and relative characteristics; configuring critical factors identified through analyzing the determined unique features; confirming the critical factors is valid through experimental studies; and determining critical factors that are based on the confirmed experimental studies.

According to one embodiment, the identified unique features are amino acid length, molecular weight, pI value, bending potential, rigidity, flexibility, structural feature, hydropathy, residue structure, amino acid composition and secondary structure.

According to one embodiment, the determined six critical factors consist of the following characteristics:

a. Amino Acid Length: 9 to 13

b. Bending Potential: Proline (P) positioned in the middle (i.e., U5, U6, U7, or U8) and at the end of the sequence.

c. Rigidity/Flexibility: Instability Index (II): 40 to 60

d. Structural Feature: Aliphatic Index (AI): 180 to 220

e. Hydropathy: GRAVY: 2.1 to 2.6.

f. Amino Acid Composition: All of composed amino acids are hydrophobic and aliphatic amino acids (A, V, L, I and P)

G. Secondary structure: α-Helix

The present disclosure further provides a method of developing the aMTD sequences.

The method comprises designing a platform of aMTDs having the below general formula described below; [General formula] X1-X2-X3-X4-U5-U6-U7-U8-X9-X10-X11-P

wherein (P) at the end of sequence (12′) is proline, one of U sites is proline, X(s) and U(s) which is not proline are A, V, L and/or I; and confirming whether a designed amino acid sequence satisfy six critical factors as follows:

a. Amino Acid Length: 9 to 13

b. Bending Potential: Proline (P) positioned in the middle (i.e., U5, U6, U7, or U8) and at the end of the sequence.

c. Rigidity/Flexibility: Instability Index (II): 40 to 60

d. Structural Feature: Aliphatic Index (AL): 180 to 220

e. Hydropathy: GRAVY: 2.1 to 2.6.

f. Amino Acid Composition: All of composed amino acids are hydrophobic and aliphatic amino acids (A, V, L, I and P)

According to one embodiment, the six critical factors obtained the method of identifying unique features of aMTDs consist of the following factors:

a. Amino Acid Sequence: 12

b. Bending Potential: Proline (P) is positioned in the middle (i.e., U5, U6, U7, or U8) and at the end (12′) of the sequence.

c. Rigidity/Flexibility: Instability Index (II): 41.3 to 57.3

d. Structural Feature: Aliphatic Index (AI): 187.5 to 220

e. Hydropathy: GRAVY: 2.2 to 2.6.

f. Amino Acid Composition: All of composed amino acids are hydrophobic and aliphatic amino acids (A, V, L, I and P) According to one embodiment, the secondary structure of the aMTD is α-Helix.

According to one embodiment, the method further comprises developing the expression vectors of aMTD sequences fused to cargo proteins; selecting proper bacteria strain for inducible expression; purifying and preparing of aMTD-fused to cargo proteins in soluble form; and confirming their cell-permeability.

One aspect of present invention further provides isolated recombinant proteins with a cell-permeability. The isolated recombinant protein comprises an advanced macromolecule transduction domain (aMTD) sequences having amino acid sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 240; and a biologically active molecule.

According to one embodiment, the biologically active molecules are any one selected from the group consisting of growth factors, enzymes, transcription factors, toxins, antigenic peptides, antibodies and antibody fragments.

According to one embodiment, the biologically active molecules are any one selected from the group consisting of enzymes, hormones, carriers, immunoglobulins, antibodies, structural proteins, motor functioning peptides, receptors, signaling peptides, storing peptides, membrane peptides, transmembrane peptides, internal peptides, external peptides, secreting peptides, virus peptides, native peptides, glycated proteins, fragmented proteins, disulfide bonded proteins, recombinant proteins, chemically modified proteins and prions.

According to one embodiment, the biologically active molecules are any one selected from the group consisting of nucleic acids, coding nucleic acid sequences, mRNAs, antisense RNA molecules, carbohydrates, lipids and glycolipids.

According to one embodiment, the biologically active molecules are at least one selected from the group consisting of biotherapeutic chemicals and toxic chemicals.

One aspect of the present invention further provides a method of genetically or epigenetically engineering and/or modifying biologically active molecules to have a cell-permeability. The method comprises fusing aMTDs to biologically active molecules under the optimized and effective conditions to generate biologically active molecules that can be cell-permeable, wherein the aMTD consists of any one of amino acid sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 240.

One aspect of the present invention also pertains to cell-permeable recombinant protein for generation of induced pluripotent stem cells (iPSCs) based on advanced macromolecule transduction domain (aMTD) sequences capable of mediating the transduction of biologically active macromolecules into live cells.

Other aspect of the present invention relates to cell-permeable protein-based generation of induced pluripotent stem cells (iPSCs) based on an efficient use of aMTD sequences for peptide delivery, protein delivery and intracellular protein delivery.

One aspect of the present invention provides an iCP-RF (improved Cell-Permeable Reprogramming Factor) recombinant protein, which comprises a RF protein selected from the group consisting of OCT4, SOX2, CMYC, KLF4, NANOG, LIN28 and ZSCAN4, and an advanced macromolecule transduction domain (aMTD) being composed of 9 to 13 amino acid sequences and having improved cell or tissue permeability, wherein the aMTD is fused to one end or both ends of the RF protein and has the following features of:

(a) being composed of 3 or more amino acids selected from the group consisting of Ala, Val, Ile, Leu, and Pro;

(b) having proline as amino acids corresponding to any one or more of positions 5 to 8, and 12 of its amino acid sequence; and

(c) having an instability index of 40 to 60; an aliphatic index of 180 to 220; and a grand average of hydropathy (GRAVY) of 2.1 to 2.6, as measured by Protparam.

According to one embodiment, one or more solubilization domain (SD)(s) are further fused to the end(s) of one or more of the RF protein and the aMTD.

According to another embodiment, the aMTD may have α-Helix structure. According to still another embodiment, the aMTD may be composed of 12 amino acid sequences and represented by the following general formula: [General formula] X1-X2-X3-X4-U5-U6-U7-U8-X9-X10-X11-P

wherein X(s) refers to Alanine (A), Valine (V), Leucine (L) or Isoleucine (I); one of U refers to proline (P) and the other U(s) refer to A, V, L or I; and P refers to proline.

Another aspect of the present invention provides an iCP-RF recombinant protein which is represented by any one of the following structural formula: A-B—C and A-C—B—C

wherein A is an advanced macromolecule transduction domain (aMTD) having improved cell or tissue permeability, B is a RF protein selected from the group consisting of OCT4, SOX2, CMYC, KLF4, NANOG, LIN28 and ZSCAN4, and C is a solubilization domain (SD); and

the aMTD is composed of 9 to 13 amino acid sequences and has the following features of:

(a) being composed of 3 or more amino acids selected from the group consisting of Ala, Val, Ile, Leu, and Pro;

(b) having proline as amino acids corresponding to any one or more of positions 5 to 8, and 12 of its amino acid sequence;

(c) having an instability index of 40 to 60; an aliphatic index of 180 to 220; and a grand average of hydropathy (GRAVY) of 2.1 to 2.6, as measured by Protparam; and

(d) having α-Helix structure.

According to one embodiment of the present invention, the SD(s) are one or more selected from the group consisting of SDA, SDB, SDB′, SDC, SDD, SDE and SDF.

According to one embodiment of the present invention, the RF protein may have an amino acid sequence of SEQ ID NOs: 816 to 822.

According to another embodiment of the present invention, the RF protein may be encoded by a polynucleotide sequence of SEQ ID NOs: 823 to 829.

According to still another embodiment of the present invention, the RF protein may further include a ligand selectively binding to a receptor of a cell, a tissue, or an organ.

According to still another embodiment of the present invention, the aMTD may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 240.

According to still another embodiment of the present invention, the aMTD may be encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 241 to 480.

According to still another embodiment of the present invention, the SD(s), independently, may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 798 to 804.

According to still another embodiment of the present invention, the SD(s), independently, may be encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 805 to 811.

According to still another embodiment of the present invention, the RF recombinant protein may have one or more selected from a histidine-tag affinity domain and a nuclear localization sequence (NLS) additionally fused to one end thereof.

According to still another embodiment of the present invention, the histidine-tag affinity domain may have an amino acid sequence of SEQ ID NO: 812, and the NLS may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 814 and 872.

According to still another embodiment of the present invention, the histidine-tag affinity domain may be encoded by a polynucleotide sequence of SEQ ID NO: 813, and the NLS may be encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 815 and 873.

According to still another embodiment of the present invention, the fusion may be formed via a peptide bond or a chemical bond.

According to still another embodiment of the present invention, the iCP-RF recombinant protein may be used for generation of induced pluripotent stem cells (iPSCs) from somatic cells.

Still another aspect of the present invention provides a polynucleotide sequence encoding the iCP-RF recombinant protein.

According to one embodiment of the present invention, the polynucleotide sequence may be selected from the group consisting of SEQ ID NOs: 831, 837, 843, 849, 855, 861 and 867.

According to another embodiment of the present invention, the polynucleotide sequence may be selected from the group consisting of SEQ ID NOs: 833, 839, 845, 851, 857, 863 and 869.

Still another aspect of the present invention provides a recombinant expression vector including the polynucleotide sequence.

Still another aspect of the present invention provides a transformant transformed with the recombinant expression vector.

Still another aspect of the present invention provides a preparing method of the iCP-RF recombinant protein including preparing the recombinant expression vector; preparing the transformant using the recombinant expression vector; culturing the transformant; and recovering the recombinant protein expressed by the culturing.

Still another aspect of the present invention provides a composition including the iCP-RF recombinant protein as an active ingredient.

According to one embodiment of the present invention, the composition generates induced pluripotent stem cells (iPSCs) from somatic cells.

Still another aspect of the present invention provides use of the iCP-RF recombinant protein for generating iPSCs from somatic cells.

Still another aspect of the present invention provides a method of generating iPSCs from somatic cells, including preparing somatic cells; and treating the somatic cells with an effective amount of the iCP-RF recombinant protein.

In one embodiment of the present invention, the somatic cells may be derived from a mammal.

Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Although a certain method and a material is described herein, it should not be construed as being limited thereto, any similar or equivalent method and material to those may also be used in the practice or testing of the present invention. All publications mentioned herein are incorporated herein by reference in their entirety to disclose and describe the methods and/or materials in connection with which the publications are cited. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

A “peptide” refers to a chain-type polymer formed by amino acid residues which are linked to each other via peptide bonds, and used interchangeably with “polypeptide.” Further, a “polypeptide” includes a peptide and a protein.

Further, the term “peptide” includes amino acid sequences that are conservative variations of those peptides specifically exemplified herein. The term “conservative variation,” as used herein, denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include substitution of one hydrophobic residue, such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine, or methionine for another, or substitution of one polar residue for another, for example, substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like. Neutral hydrophilic amino acids which may be substituted for one another include asparagine, glutamine, serine, and threonine.

The term “conservative variation” also includes use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibodies raised to the substituted polypeptide also immunoreacts with the unsubstituted polypeptide. Such conservative substitutions are within the definition of the classes of the peptides according to one embodiment of the present invention.

A person having ordinary skill in the art may make similar substitutions to obtain peptides having higher cell permeability and a broader host range. For example, one embodiment of the present invention provides peptides corresponding to amino acid sequences (e.g. SEQ ID NOs: 1 to 240) provided herein, as well as analogues, homologs, isomers, derivatives, amidated variations, and conservative variations thereof, as long as the cell permeability of the peptide remains.

Minor modifications to primary amino acid sequence of the peptides according to one embodiment of the present invention may result in peptides which have substantially equivalent or enhanced cell permeability, as compared to the specific peptides described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous.

All peptides may be synthesized using L-amino acids, but D forms of all of the peptides may be synthetically produced. In addition, C-terminal derivatives, such as C-terminal methyl esters and C-terminal amidates, may be produced in order to increase the cell permeability of the peptide according to one embodiment of the present invention.

All of the peptides produced by these modifications are included herein, as long as in the case of amidated versions of the peptide, the cell permeability of the original peptide is altered or enhanced such that the amidated peptide is therapeutically useful. It is envisioned that such modifications are useful for altering or enhancing cell permeability of a particular peptide.

Furthermore, deletion of one or more amino acids may also result in a modification to the structure of the resultant molecule without any significant change in its cell permeability. This may lead to the development of a smaller active molecule which may also have utility. For example, amino- or carboxyl-terminal amino acids which may not be required for the cell permeability of a particular peptide may be removed.

The term “gene” refers to an arbitrary nucleic acid sequence or a part thereof having a functional role in protein coding or transcription, or regulation of other gene expression. The gene may be composed of all nucleic acids encoding a functional protein or a part of the nucleic acid encoding or expressing the protein. The nucleic acid sequence may include a gene mutation in exon, intron, initiation or termination region, promoter sequence, other regulatory sequence, or a unique sequence adjacent to the gene.

The term “primer” refers to an oligonucleotide sequence that hybridizes to a complementary RNA or DNA target polynucleotide and serves as the starting points for the stepwise synthesis of a polynucleotide from mononucleotides by the action of a nucleotidyltransferase as occurs, for example, in a polymerase chain reaction.

The term “coding region” or “coding sequence” refers to a nucleic acid sequence, a complement thereof, or a part thereof which encodes a particular gene product or a fragment thereof for which expression is desired, according to the normal base pairing and codon usage relationships. Coding sequences include exons in genomic DNA or immature primary RNA transcripts, which are joined together by the cellular biochemical machinery to provide a mature mRNA. The anti-sense strand is the complement of the nucleic acid, and the coding sequence may be deduced therefrom.

One aspect of the present invention provides an iCP-RF recombinant protein, which comprises a RF protein selected from the group consisting of OCT4, SOX2, CMYC, KLF4, NANOG, LIN28 and ZSCAN4, and an advanced macromolecule transduction domain (aMTD) being composed of 9 to 13 amino acid sequences, preferably 10 to 12 amino acid sequences, and having improved cell or tissue permeability, wherein the aMTD is fused to one end or both ends of the RF protein and has the following features of:

(a) being preferably composed of 3 or more amino acids selected from the group consisting of Ala, Val, Ile, Leu, and Pro;

(b) having proline as amino acid sequences corresponding to any one or more of positions 5 to 8, and 12 of its amino acids, and preferably one or more of positions 5 to 8 and position 12 of its amino acid sequence; and

(c) having an instability index of preferably 40 to 60 and more preferably 41-58; an aliphatic index of preferably 180 to 220 and more preferably 185 to 225; and a grand average of hydropathy (GRAVY) of preferably 2.1 to 2.6 and more preferably 2.2 to 2.6 as measured by Protparam (see web.expasy.org).

To determine the GRAVY value of the protein analysed easily, the ProtParam (Gasteiger E. et al., Protein Identification and Analysis Tools on the ExPASy Server, J M Walker ed., The Proteomics Protocols Handbook, Humana Press, 2005, 571-607) program is used. ProtParam program (web.expasy.org) is a computational formula which provides various physicochemical properties of the proteins studied by analysing their sequence; when the said amino-acid sequence is entered, the program calculates the GRAVY value of the protein whose degree of hydrophobicity is to be measured.

According to one embodiment, one or more solubilization domain (SD)(s) are further fused to one or more of the RF protein and the aMTD, preferably one end or both ends of the RF protein, and more preferably the C-terminus or both the C-terminus and the N-terminus of the RF protein.

According to another embodiment, the aMTD may have α-Helix structure.

According to still another embodiment, the aMTD may be preferably composed of 12 amino acid sequences and represented by the following general formula: [General formula] X1-X2-X3-X4-U5-U6-U7-U8-X9-X10-X11-P

wherein X(s) refers to Alanine (A), Valine (V), Leucine (L) or Isoleucine (I); one of U refers to proline (P) and the other U(s) refer to A, V, L or I; and P refers to proline.

Still another aspect of the present invention provides an iCP-RF recombinant protein which is represented by any one of structural formula A-B—C and/or A-C—B—C, and preferably by A-B—C for iCP-OCT4, iCP-CMYC, iCP-NANOG, iCP-LIN28 or iCP-ZSCAN4 recombinant protein and by A-C—B—C for iCP-SOX2, iCP-KLF4 recombinant protein: wherein A is an advanced macromolecule transduction domain (aMTD) having improved cell or tissue permeability, B is a RF protein selected from the group consisting of OCT4, SOX2, CMYC, KLF4, NANOG, LIN28 and ZSCAN4, and C is a solubilization domain (SD); and

the aMTD is composed of 9 to 13, preferably 10 to 12 amino acid sequences and has the following features of:

(a) being composed of 3 or more amino acids selected from the group consisting of Ala, Val, Ile, Leu, and Pro;

(b) having proline as amino acids corresponding to any one or more of positions 5 to 8, and 12 of its amino acid sequence, and preferably, one or more of positions 5 to 8 and position 12 of its amino acid sequence;

(c) having an instability index of preferably 40 to 60 and more preferably 41 to 58; an aliphatic index of preferably 180 to 220 and more preferably 185 to 225; and a grand average of hydropathy (GRAVY) of preferably 2.1 to 2.6 and more preferably 2.2 to 2.6, as measured by Protparam (see web.expasy.org); and

(d) preferably having α-Helix structure.

Preferably, the iCP-RF recombinant proteins may be iCP-OCT4, iCP-SOX2, iCP-KLF4, iCP-CMYC, iCP-NANOG, iCP-LIN28 or iCP-ZSCAN4.

In one embodiment of the present invention, the SD(s) may has one or more selected from the group consisting of SDA, SDB, SDB′, SDC, SDD, SDE and SDF, and preferably one to four selected therefrom. When the SD(s) may be two or more, they may be the same as or different from each other.

In one embodiment of the present invention, the RF protein may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 816 to 822.

In another embodiment of the present invention, the RF protein may be encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 823 to 829.

When the iCP-RF recombinant protein is intended to be delivered to a particular cell, tissue, or organ, the RF protein may form a fusion product, together with an extracellular domain of a ligand capable of selectively binding to a receptor which is specifically expressed on the particular cell, tissue, or organ, or monoclonal antibody (mAb) capable of specifically binding to the receptor or the ligand and a modified form thereof.

The binding of the peptide and a biologically active substance may be formed either by indirect linkage by a cloning technique using an expression vector at a nucleotide level or by direct linkage via chemical or physical covalent or non-covalent bond of the peptide and the biologically active substance.

In still another embodiment of the present invention, the RF protein may preferably further include a ligand selectively binding to a receptor of a cell, a tissue, or an organ.

In one embodiment of the present invention, the aMTD may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 240. The aMTD may be preferably aMTD₁₆₁ of SEQ ID NO: 39, aMTD₁₆₅ of SEQ ID NO: 43, aMTD₃₆₃ of SEQ ID NO: 84, aMTD₄₀₅ of SEQ ID NO: 96, aMTD₅₆₃ of SEQ ID NO: 131, aMTD₈₈₉ of SEQ ID NO: 223, aMTD₈₉₅ of SEQ ID NO: 226 or aMTD₉₀₄ of SEQ ID NO: 233, and more preferably aMTD₁₆₁ of SEQ ID NO: 39 or aMTD₅₆₃ of SEQ ID NO: 131.

In still another embodiment of the present invention, the aMTD may be encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 241 to 480. The aMTD may be preferably aMTD₁₆₁ encoded by a polynucleotide sequence of SEQ ID NO: 279, aMTD₁₆₅ encoded by a polynucleotide sequence of SEQ ID NO: 283, aMTD₃₆₃ encoded by a polynucleotide sequence of SEQ ID NO: 324, aMTD₄₀₅ encoded by a polynucleotide sequence of SEQ ID NO: 336, aMTD₅₆₃ encoded by a polynucleotide sequence of SEQ ID NO: 371, aMTD₈₈₉ encoded by a polynucleotide sequence of SEQ ID NO: 463, aMTD₈₉₅ encoded by a polynucleotide sequence of SEQ ID NO: 466 or aMTD₉₀₄ encoded by a polynucleotide sequence of SEQ ID NO: 473, and more preferably preferably aMTD₁₆₁ encoded by a polynucleotide sequence of SEQ ID NO: 279 or aMTD₅₆₃ encoded by a polynucleotide sequence of SEQ ID NO: 371.

In still another embodiment of the present invention, the SD(s) may have an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 798 to 804. The SD may be preferably SDA of SEQ ID NO: 798 and/or SDB of SEQ ID NO: 799, and more preferably SDB of SEQ ID NO: 799 or both SDA of SEQ ID NO: 798 and SDB of SEQ ID NO: 799 which have superior structural stability.

In still another embodiment of the present invention, the SDs may be encoded by a polynucleotide sequence independently selected from the group consisting of SEQ ID NOs: 805 to 811. The SD may be preferably SDA encoded by a polynucleotide sequence of SEQ ID NO: 805 or SDB encoded by a polynucleotide sequence of SEQ ID NO: 806, and more preferably SDB or both SDA and SDB having superior structural stability, which is encoded by a polynucleotide sequence of SEQ ID NOs: 805 and 806.

In still another embodiment of the present invention, the iCP-RF recombinant protein may be preferably selected from the group consisting of:

1) a recombinant protein, in which RF protein having an amino acid sequence of SEQ ID NOs: 816 to 822 is fused to the N-terminus or the C-terminus of aMTD having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 240, preferably SEQ ID NOs: 39, 43, 84, 96, 131, 223, 226 and 233, and more preferably SEQ ID NO: 39 and 131;

2) a recombinant protein, in which SD having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 798 to 804, preferably SEQ ID NOs: 798, 799, 801, 802, 803, and 804, and more preferably SEQ ID NO: 798 and 799 is further fused to the N-terminus or the C-terminus of the RF protein in the recombinant protein of 1); and

3) a recombinant protein, in which one or more of a histidine tag having an amino acid sequence of SEQ ID NO: 812 and a NLS may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 814 and 872 are further fused to the N-terminus or the C-terminus of the aMTD in the recombinant protein of 1) or 2).

When the RF protein may be delivered to terminally differentiated somatic cells, it may reprogram the somatic cells to induced pluripotent stem cells (iPSCs). The recombinant expression vector may include a tag sequence which makes it easy to purify the recombinant protein, for example, consecutive histidine codon, maltose binding protein codon, Myc codon, etc., and further include a fusion partner to enhance solubility of the recombinant protein, etc. Further, for the overall structural and functional stability of the recombinant protein or flexibility of the proteins encoded by respective genes, the recombinant expression vector may further include one or more glycine, proline, and spacer amino acid or polynucleotide sequences including AAY amino acids. Furthermore, the recombinant expression vector may include a sequence specifically digested by an enzyme in order to remove an unnecessary region of the recombinant protein, an expression regulatory sequence, and a marker or reporter gene sequence to verify intracellular delivery, but is not limited thereto.

In still another embodiment of the present invention, the iCP-RF recombinant protein may preferably have a one or more of a histidine-tag affinity domain and a nuclear localization sequence (NLS) additionally fused to one end thereof. Preferably, the histidine-tag or the NLS may be fused to the N-terminus of the RF protein, and more preferably, both of the histidine-tag and the NLS may be fused to the N-terminus of the RF protein.

In still another embodiment of the present invention, the histidine-tag affinity domain may have an amino acid sequence of SEQ ID NO: 812, and the NLS may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 814 and 872. The NLS may have one selected from the group consisting of NLS-1 and NLS-2.

In still another embodiment of the present invention, the histidine-tag affinity domain may be encoded by a polynucleotide sequence of SEQ ID NO: 813, and the NLS may be encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 815 and 873.

In still another embodiment of the present invention, the fusion may be formed via a peptide bond or a chemical bond.

The chemical bond may be preferably selected from the group consisting of disulfide bonds, diamine bonds, sulfide-amine bonds, carboxyl-amine bonds, ester bonds, and covalent bonds.

According to still another embodiment of the present invention, the iCP-RF recombinant protein may be used for generation to induced pluripotent stem cells (iPSCs) from somatic cells.

The induced pluripotent stem cells (iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from adult cells; terminally differentiated somatic cells. The iPSCs are typically derived by introducing products of specific set of pluripotency-associated genes, or “reprogramming factors,” into a given cell type. The reprogramming factors include OCT4 (Octamer-binding transcription factor 4), SOX2 (Sex determining region Y-box 2), NANOG (Homeobox protein NANOG), CMYC (c-Myc), KLF4 (Kruppel-like factor 4), LIN28 (Lin-28 homolog A) and ZSCAN4 (Zinc finger and SCAN domain containing 4). The OCT4, SOX2 and NANOG are transcription factors that require for the maintenance of embryonic stem cells in pluripotent status, the CMYC, KLF4 and LIN28 are intranuclear proteins that facilitate self-renewal and inhibit differentiation of cells, and ZSCAN4 is a protein involved in telomere elongation and genome stabilization. The somatic cells form mouse or human can be reprogrammed to the pluripotent state via viral transduction with the sets of reprogramming factors. While this combination is most conventional in producing iPSCs, each of the factors can be functionally replaced by related transcription factors, miRNAs, small molecules, or even non-related genes such as lineage specifiers. The iPSC derivation is typically a slow and inefficient process, taking 1 to 2 weeks/mouse cells and 3 to 4 weeks/human cells, with efficiencies around 0.01% to 0.1%. However, considerable advances have been made in improving the efficiency and the time it takes to obtain iPSCs. Upon introduction of reprogramming factors (RFs), cells begin to form colonies that resemble pluripotent stem cells, which can be isolated based on their morphology, conditions that select for their growth, or through expression of surface markers (alkaline phosphatase, OCT4, TRA-1-60, TRA-1-81, etc.) or reporter genes.

Preferably, the iCP-RF recombinant proteins may be iCP-OCT4, iCP-SOX2, iCP-KLF4, iCP-CMYC, iCP-NANOG, iCP-LIN28 or iCP-ZSCAN4.

Still another aspect of the present invention provides a polynucleotide sequence encoding the iCP-RF recombinant protein.

The polynucleotide sequence according to one embodiment of the present invention may be present in a vector in which the polynucleotide sequence is operably linked to regulatory sequences capable of providing for the expression of the polynucleotide sequence by a suitable host cell.

According to one embodiment of the present invention, the polynucleotide sequence may be selected from the following groups:

1) a polynucleotide sequence, in which any one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 241 to 480, preferably SEQ ID NOs: 279, 283, 324, 336, 371, 463, 466 and 473, and more preferably SEQ ID NOs: 279 and 371, is operably linked with and a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 823 to 829; and

2) a polynucleotide sequence, in which any one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 831, 833, 835, 837, 839, 841, 843, preferably SEQ ID NOs: 805, 806, 808, 809, 810, and 811, and more preferably SEQ ID NOs: 805 and 806 is further operably linked to the polynucleotide sequence of 1).

Within the expression vector, the term “operably linked” is intended to mean that the polynucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the polynucleotide sequence. The term “regulatory sequence” is intended to include promoters, enhancers, and other expression control elements. Such operable linkage with the expression vector can be achieved by conventional gene recombination techniques known in the art, while site-directed DNA cleavage and linkage are carried out by using conventional enzymes known in the art.

The expression vectors may contain a signal sequence or a leader sequence for membrane targeting or secretion, as well as regulatory sequences such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, an enhancer and the like. The promoter may be a constitutive or an inducible promoter. Further, the expression vector may include one or more selectable marker genes for selecting the host cell containing the expression vector, and may further include a polynucleotide sequence that enables the vector to replicate in the host cell in question.

The expression vector constructed according to the present invention may be the vector where the polynucleotide encoding the iCP-RF recombinant protein (where an aMTD is fused to the N-terminus or C-terminus of a RF protein) is inserted within the multiple cloning sites (MCS), preferably NdeI/SalI site of a pET-28a(+) vector (Novagen, USA).

In still another embodiment of the present invention, the polynucleotide encoding the SD being additionally fused to the N-terminus or C-terminus of a RF protein may be inserted into a cleavage site of restriction enzyme (NdeI, EcoRI, SalI, XhoI, etc.) within the multiple cloning sites (MCS) of a pET-28a(+) vector (Novagen, USA).

In still another embodiment of the present invention, the polynucleotide is cloned into a pET-28a(+) vector bearing a NLS residues to the N-terminus of the iCP-RF recombinant protein to allow efficient nuclear transport.

In still another embodiment of the present invention, the polynucleotide is cloned into a pET-28a(+) vector bearing a His-tag sequence so as to fuse six histidine residues to the N-terminus of the iCP-RF recombinant protein to allow easy purification.

According to one embodiment of the present invention, the polynucleotide sequence may be a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 831, 837, 843, 849, 855, 861 and 867.

According to another embodiment of the present invention, the polynucleotide sequence may be further fused with SD, and may be a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 833, 839, 845, 851, 857, 863 and 869.

According to still another embodiment of the present invention, the polynucleotide sequence may be fused with a histidine-tag affinity domain and NLS, and may be a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 835, 841, 847, 853, 859, 865 and 871.

Preferably, the iCP-RF recombinant protein of the present invention may be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 833, 839, 845, 851, 857, 863 and 869.

Still another aspect of the present invention provides a recombinant expression vector including the polynucleotide sequence.

Preferably, the vector may be inserted in a host cell and recombined with the host cell genome, or refers to any nucleic acid including a nucleotide sequence competent to replicate spontaneously as an episome. Such a vector may include a linear nucleic acid, a plasmid, a phagemid, a cosmid, an RNA vector, a viral vector, etc.

Preferably, the vector may be genetically engineered to incorporate the nucleic acid sequence encoding the recombinant protein in an orientation either N-terminal and/or C-terminal to a nucleic acid sequence encoding a peptide, a polypeptide, a protein domain, or a full-length protein of interest, and in the correct reading frame so that the recombinant protein consisting of aMTD, RF protein, and preferably SD may be expressed. Expression vectors may be selected from those readily available for use in prokaryotic or eukaryotic expression systems.

Standard recombinant nucleic acid methods may be used to express a genetically engineered recombinant protein. The nucleic acid sequence encoding the recombinant protein according to one embodiment of the present invention may be cloned into a nucleic acid expression vector, e.g., with appropriate signal and processing sequences and regulatory sequences for transcription and translation, and the protein may be synthesized using automated organic synthetic methods. Synthetic methods of producing proteins are described in, for example, the literature [Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis by Gregg B. Fields (Editor), Sidney P. Colowick, Melvin I. Simon (Editor), Academic Press (1997)].

In order to obtain high level expression of a cloned gene or nucleic acid, for example, a cDNA encoding the recombinant protein according to one embodiment of the present invention, the recombinant protein sequence may be typically subcloned into an expression vector that includes a strong promoter for directing transcription, a transcription/translation terminator, and in the case of a nucleic acid encoding a protein, a ribosome binding site for translational initiation. Suitable bacterial promoters are well known in the art and are described, e.g., in the literature [Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3d Edition, Cold Spring Harbor Laboratory, N.Y. (2001); and Ausube, et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y. (1989)]. Bacterial expression systems for expression of the recombinant protein according to one embodiment of the present invention are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene 22: 229-235 (1983); Mosbach et al., Nature 302: 543-545 (1983)). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. The eukaryotic expression vector may be preferably an adenoviral vector, an adeno-associated vector, or a retroviral vector.

Generally, the expression vector for expressing the cell permeable recombinant protein according to one embodiment of present invention in which the cargo protein, i.e. RF protein, is attached to the N-terminus, C-terminus, or both termini of aMTD may include regulatory sequences including, for example, a promoter, operably attached to a sequence encoding the advanced macromolecule transduction domain. Non-limiting examples of inducible promoters that may be used include steroid-hormone responsive promoters (e.g., ecdysone-responsive, estrogen-responsive, and glutacorticoid-responsive promoters), tetracycline “Tet-On” and “Tet-Off” systems, and metal-responsive promoters.

The recombinant protein may be introduced into an appropriate host cell, e.g., a bacterial cell, a yeast cell, an insect cell, or a tissue culture cell. The recombinant protein may also be introduced into embryonic stem cells in order to generate a transgenic organism. Large numbers of suitable vectors and promoters are known to those skilled in the art and are commercially available for generating the recombinant protein.

Known methods may be used to construct vectors including the polynucleotide sequence according to one embodiment of the present invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic recombination. For example, these techniques are described in the literatures [Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3d Edition, Cold Spring Harbor Laboratory, N. Y. (2001); and Ausubel et al., Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience, N.Y. (1989)].

Still another aspect of the present invention provides a transformant transformed with the recombinant expression vector.

The transformation includes transfection, and refers to a process whereby a foreign (extracellular) DNA, with or without an accompanying material, enters into a host cell. The “transfected cell” refers to a cell into which the foreign DNA is introduced into the cell, and thus the cell harbors the foreign DNA. The DNA may be introduced into the cell so that a nucleic acid thereof may be integrated into the chromosome or replicable as an extrachromosomal element. The cell introduced with the foreign DNA, etc. is called a transformant.

As used herein, ‘introducing’ of a protein, a peptide, an organic compound into a cell may be used interchangeably with the expression of ‘carrying,’ ‘penetrating,’ ‘transporting,’ ‘delivering,’ ‘permeating’ or ‘passing.’

It is understood that the host cell refers to a eukaryotic or prokaryotic cell into which one or more DNAs or vectors are introduced, and refers not only to the particular subject cell but also to the progeny or potential progeny thereof. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

The host cells may be preferably bacterial cells, and as the bacterial cells, there are, in principle, no limitations. They may be eubacteria (gram-positive or gram-negative) or archaebacteria, as long as they allow genetic manipulation for insertion of a gene of interest, preferably for site-specific integration, and they may be cultured on a manufacturing scale. Preferably, the host cells may have the property to allow cultivation to high cell densities.

Examples of bacterial host cells that may be used in the preparation of the recombinant protein are E. coli (Lee, 1996; Hannig and Makrides, 1998), Bacillus subtilis, Pseudomonas fluorescens (Squires et al., 2004; Retallack et al., 2006) as well as various Corynebacterium (US 2006/0003404 A1) and Lactococcus lactis (Mierau et al., 2005) strains.

Preferably, the host cells are Escherichia coli cells.

More preferably, the host cell may include an RNA polymerase capable of binding to a promoter regulating the gene of interest. The RNA polymerase may be endogenous or exogenous to the host cell.

Preferably, host cells with a foreign strong RNA polymerase may be used. For example, Escherichia coli strains engineered to carry a foreign RNA polymerase (e.g. like in the case of using a T7 promoter a T7-like RNA polymerase in the so-called “T7 strains”) integrated in their genome may be used. Examples of T7 strains, e.g. BL21(DE3), HMS174(DE3), and their derivatives or relatives (see Novagen, pET System manual, 11^(th) edition), may be widely used and commercially available. Preferably, BL21-CodonPlus (DE3)-RIL or BL21-CodonPlus (DE3)-RIPL (Agilent Technologies) may be used. These strains are DE3 lysogens containing the T7 RNA polymerase gene under control of the lacUV5 promoter. Induction with IPTG allows production of T7 RNA polymerase which then directs the expression of the gene of interest under the control of the T7 promoter.

The host cell strains, E. coli BL21(DE3) or HMS174(DE3), which have received their genome-based T7 RNA polymerase via the phage DE3, are lysogenic. It is preferred that the T7 RNA polymerase contained in the host cell has been integrated by a method which avoids, or preferably excludes, the insertion of residual phage sequences in the host cell genome since lysogenic strains have the disadvantage to potentially exhibit lytic properties, leading to undesirable phage release and cell lysis.

Still another aspect of the present invention provides a preparing method of the iCP-RF recombinant protein including preparing the recombinant expression vector; preparing the transformant using the recombinant expression vector; culturing the transformant; and recovering the recombinant protein expressed by culturing.

Culturing may be preferably in a mode that employs the addition of a feed medium, this mode being selected from the fed-batch mode, semi-continuous mode, or continuous mode. The bacterial expression host cells may include a DNA construct which is integrated in their genome and carrying the DNA sequence encoding the protein of interest under the control of a promoter that enables expression of said protein.

There are no limitations in the type of the culture medium. The culture medium may be semi-defined, i.e. containing complex media compounds (e.g. yeast extract, soy peptone, casamino acids), or it may be chemically defined, without any complex compounds. Preferably, a defined medium may be used. The defined media (also called minimal or synthetic media) are exclusively composed of chemically defined substances, i.e. carbon sources such as glucose or glycerol, salts, vitamins, and, in view of a possible strain auxotrophy, specific amino acids or other substances such as thiamine. Most preferably, glucose may be used as a carbon source. Usually, the carbon source of the feed medium serves as the growth-limiting component which controls the specific growth rate.

Host cells may be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents. The literature [Scopes, Protein Purification: Principles and Practice, New York: Springer-Verlag (1994)] describes a number of general methods for purifying recombinant (and non-recombinant) proteins. The methods may include, e.g., ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, selective precipitation, dialysis, and hydrophobic interaction chromatography. These methods may be adapted to devise a purification strategy for the cell permeable recombinant protein. If the cell permeable recombinant protein includes a purification handle, such as an epitope tag or a metal chelating sequence, affinity chromatography may be used to easily purify the protein.

The amount of the protein produced may be evaluated by detecting the advanced macromolecule transduction domain directly (e.g., using Western analysis) or indirectly (e.g., by assaying materials derived from the cells for specific DNA binding activity, such as by electrophoretic mobility shift assay). Proteins may be detected prior to purification, during any stage of purification, or after purification. In some implementations, purification or complete purification may not be necessary.

The genetically engineered recombinant protein prepared by the method according to one embodiment of the present invention may be a cell/tissue-permeable protein. In particular, the recombinant protein may be activating or inhibiting transcription of a target gene in the nucleus to control transcription of the gene.

The cell permeable recombinant proteins according to one embodiment of present invention may be used in vitro to investigate protein function or may be used to maintain cells in a desired state.

Still another aspect of the present invention provides a composition including the iCP-RF Recombinant Protein as an active ingredient.

The composition may be induced dedifferentiation of terminally differentiated somatic cells into iPSCs. The composition may preferably comprise the active ingredient in an amount of 0.1 to 99.9% by weight, based on the total weight of the composition. The composition may comprise one or more recombinant proteins of OCT4, SOX2, CMYC, KLF4, NANOG, LIN28 and ZSCAN4. Preferably, for effective generation of iPSCs from somatic cells, the composition may include OCT4, SOX2, CMYC, KLF4 and LIN28 recombinant proteins, OCT4, SOX2, KLF4, CMYC, LIN28 and ZSCAN4 recombinant proteins, or OCT4, CMYC and NANOG recombinant proteins. In addition to the active ingredient, the composition may include a buffer, an adjuvant, etc. which is physiologically acceptable while stabilizing the recombinant protein.

Still another aspect of the present invention provides an improved cell-permeable reprogramming factor (iCP) RF recombinant protein for generating iPSCs from somatic cells.

Still another aspect of the present invention provides use of the iCP-RF recombinant protein for generating iPSCs from somatic cells.

Still another aspect of the present invention provides a method of generating iPSCs from somatic cells, including preparing terminally differentiated somatic cells; and treating the somatic cells with an effective amount of the iCP-RF recombinant protein.

The somatic cells may be derived from a mammal, and any biological cell forming the body of an organism; that is, in a brain, heart, kidney, bone, etc., any cell other than undifferentiated stem cell. In mammals, the somatic cells make up all the internal organs, skin, bones, blood and connective tissue. The somatic cells already have completed differentiation, can no more be differentiated. The terminally differentiated cells, however, can be “reprogrammed” so that they revert back to an undifferentiated pluripotent state. The reprogrammed cells are “induced pluripotent stem cells” (iPSCs) that are artificially derived from a differentiated cell, which effectively resets the genotype of the cell to that of a pluripotent state. Accordingly, the iPSCs are believed to have many features in common with natural pluripotent stem cells, such as embryonic stem cells, with regard to the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability. The iPSCs are typically derived by transfection of certain stem cell-associated genes (reprogramming factors; RFs) into non-pluripotent cells, such as adult fibroblasts.

The methods that generating iPSCs from somatic cells may be used to conveniently and efficiently establish iPSCs having pluripotency and growth ability similar to that of ES cells. The iCP-RF recombinant proteins can effectively increase the ability of reprogramming in a somatic cell, and thus can be useful in the establishment of iPSCs.

Advantageous Effects

One aspect of the present invention provides artificially constructed aMTD sequences based on the critical factors (CFs) that overcome the limitations of prior arts (MTM/MTS/MTD), such as limited diversity and unpredictable cell-permeability. Based on the CFs that assure the cell-permeability, the aMTD displays these sequences shows up to 109.9 relative fold enhanced ability compared to prior arts thereof to deliver biologically active macromolecules into live cells. Therefore, according to one aspect of the present invention, the aMTD is fused to an RF protein to provide an iCP-RF recombinant protein showing improved cell-permeability and intranuclear delivery and enhanced protein solubility and yield.

The iCP-RF recombinant proteins directly penetrate into cell membrane and transduces into nucleus with high efficiency, which can be useful to establish iPSCs from terminally differentiated somatic cells. In addition, the use of iCP-RF recombinant proteins would be safe and ethical solution to previous exogenous gene integration and provide opportunities to use patient derived-iPSCs in clinical applications.

However, the effects are not limited to the above-mentioned effects, and another effects not mentioned will be clearly understood by those skilled in the art from the following description.

DESCRIPTION OF DRAWINGS

FIG. 1 shows Structure of aMTD- or rPeptide-Fused Recombinant Proteins. A schematic diagram of the His-tagged CRA recombinant proteins is illustrated and constructed according to the present invention. The his-tag for affinity purification (white), aMTD or rPeptide (gray) and cargo A (CRA, black) are shown.

FIGS. 2a to 2c show Construction of Expression Vectors for aMTDs- or rPeptide-Fused Recombinant Proteins. These FIGs. show the agarose gel electrophoresis analysis showing plasmid DNA fragments at 645 bp insert encoding aMTDs or rPeptide-fused CRA cloned into the pET28a(+) vector according to the present invention.

FIGS. 3a to 3d show Inducible Expression of aMTD- or rPeptide-Fused Recombinant Proteins. Expressed recombinant aMTD- or random peptide-fused CRA recombinant proteins were transformed in E. coli BL21 (DE3) strain. Expression of recombinant proteins in E. coli before (−) and after (+) induction with IPTG was monitored by SDS-PAGE, and stained with Coomassie blue.

FIGS. 4a and 4b show Purification of aMTD- or rPeptide-Fused Recombinant Proteins. Expressed recombinant proteins were purified by Ni²+ affinity chromatography under the natural condition. Purification of recombinant proteins displayed through SDS-PAGE analysis.

FIGS. 5a to 5u show Determination of aMTD-Mediated Cell-Permeability. Cell-permeability of a negative control (A: rP38) and reference hydrophobic CPPs (MTM12 and MTD85) are shown. The cell-permeability of each aMTD and/or rPeptide is visually compared to that of the cargo protein lacking peptide sequence (HCA). Gray shaded area represents untreated RAW 264.7 cells (vehicle); thin light gray line represents the cells treated with equal molar concentration of FITC (FITC only); dark thick line indicates the cells treated with FITC-his-tagged CRA protein (HCA); and the cells treated with the FITC-proteins (HMCA) fused to negative control (rP38), reference CPP (MTM12 or MTD85) or new hydrophobic CPP (aMTD) are shown with light thick line and indicated by arrows.

FIGS. 6a to 6c show Determination of rPeptide-Mediated Cell-Permeability. The cell-permeability of each aMTD and/or rPeptide was visually compared to that of the cargo protein lacking peptide sequence (HCA). Gray shaded area represents untreated RAW 264.7 cells (vehicle); thin light gray line represents the cells treated with equal molar concentration of FITC (FITC only); dark thick line indicates the cells treated with FITC-his-tagged CRA protein (HCA); and the cells treated with the FITC-proteins fused to rPeptides are shown with light thick line and indicated by arrows.

FIGS. 7a to 7k shows Visualized Cell-Permeability of aMTD-Fused Recombinant Proteins. NIH3T3 cells were treated with FITC-labeled protein (10 uM) fused to aMTD for 1 hour at 37° C. Cell-permeability of the proteins was visualized by laser scanning confocal microscopy (LSM700 version).

FIG. 8 shows Visualized Cell-Permeability of rPeptide-Fused Recombinant Proteins. Cell-permeability of rPeptide-fused recombinant proteins was visualized by laser scanning confocal microscopy (LSM700 version).

FIGS. 9a to 9c show Relative Cell-Permeability of aMTD-Fused Recombinant Proteins Compared to Negative Control (rP38). The FIG. shows graphs comparing the cell-permeability of the recombinant proteins fused to aMTDs and a negative control (A: rP38).

FIGS. 10a to 10c show Relative Cell-Permeability of aMTD-Fused Recombinant Proteins Compared to Reference CPP (MTM12). The FIG. shows graphs comparing the cell-permeability of the recombinant proteins fused to aMTDs and a reference CPP (MTM12).

FIGS. 11a to 11c show Relative Cell-Permeability of aMTD-Fused Recombinant Proteins Compared to Reference CPP (MTD85). The FIG. shows graphs comparing the cell-permeability of the recombinant proteins fused to aMTDs and a reference CPP (MTD85).

FIG. 12 shows Relative Cell-Permeability of rPeptide-Mediated Recombinant Proteins Compared to Average that of aMTDs. The FIG. shows graphs comparing the cell-permeability of the recombinant proteins fused to rPeptides and that (average value: aMTD AVE) of aMTDs.

FIGS. 13a to 13d show Association of Cell-Permeability with Amino Acid Composition in aMTD Sequences. These graphs display delivery potential (Geometric Mean) of aMTDs influenced with amino acid composition (A, I, V and L).

FIGS. 14a to 14d show Association of Cell-Permeability with Critical Factors in aMTDs. These graphs show the association of cell-permeability with critical factors [bending potential: proline position (PP), rigidity/flexibility: instability index (II), structural feature: aliphatic index (AI) and hydropathy: grand average of hydropathy (GRAVY)].

FIGS. 15a to 15d show Relative Relevance of aMTD-Mediated Cell-Permeability with Critical Factors. Cell-permeability of 10 high and 10 low ranked aMTDs in their delivery potential were examined for their association with the critical factors [bending potential: proline position (PP), rigidity/flexibility: instability index (II), structural feature: aliphatic index (AI) and hydropathy: grand average of hydropathy (GRAVY)].

FIG. 16 shows Relative Relevance of rPeptide-Mediated Cell-Permeability with Hydropathy Range (GRAVY). This graph and a chart illustrate relative relevance of rPeptide-mediated cell-permeability with its hydropathy range (GRAVY).

FIG. 17a to 17g show agarose gel electrophoresis analysis showing plasmid DNA fragments insert encoding aMTD/SD-fused RF cloned into the pET28a (+) vector according to Example <6-1>.

FIG. 18 shows structure of OCT4 recombinant proteins.

FIG. 19 shows expression, purification and the solubility/yield of OCT4 recombinant protein according to Example <7-1>.

FIG. 20 shows structure of SOX2 recombinant proteins.

FIG. 21 shows expression, purification and the solubility/yield of SOX2 recombinant protein according to Example <7-2>.

FIG. 22 shows structure of KLF4 recombinant proteins.

FIG. 23 shows expression, purification and the solubility/yield of KLF4 recombinant protein according to Example <7-3>.

FIG. 24 shows structure of CMYC recombinant proteins.

FIG. 25 shows expression, purification and the solubility/yield of CMYC recombinant protein according to Example <7-4>.

FIG. 26 shows structure of NANOG recombinant proteins.

FIG. 27 shows expression, purification and the solubility/yield of NANOG recombinant protein according to Example <7-5>.

FIG. 28 shows structure of LIN28 recombinant proteins.

FIG. 29 shows expression, purification and the solubility/yield of LIN28 recombinant protein according to Example <7-6>.

FIG. 30 shows structure of ZSCAN4 recombinant proteins.

FIG. 31 shows expression, purification and the solubility/yield of ZSCAN4 recombinant protein according to Example <7-7>.

FIG. 32 shows structure of SOX2 recombinant proteins fused to 7 different aMTDs.

FIG. 33 shows expression, purification and the solubility/yield of SOX2 Recombinant Proteins Fused to 7 Different aMTDs according to Example <8-1>.

FIG. 34 shows structure of NANOG recombinant proteins fused to 5 different aMTDs.

FIG. 35 shows expression, purification and the solubility/yield of NANOG recombinant proteins fused to 5 different aMTDs according to Example <8-2>.

FIG. 36 shows structure of OCT4 recombinant proteins fused to 7 different aMTDs.

FIG. 37 shows structure of CMYC recombinant proteins fused to 8 different aMTDs.

FIG. 38 shows structure of LIN28 recombinant proteins fused to 4 different aMTDs.

FIG. 39 shows aMTD-mediated cell-permeability of RF recombinant proteins

FIG. 40 shows aMTD-mediated intracellular delivery and localization of RF recombinant proteins.

FIG. 41a shows structure of a luciferase vector having promoters of OCT4, SOX2, CMYC, KLF4, NANOG and LIN28.

FIG. 41b shows induction of transactivation with iCP-OCT4 recombinant protein in luciferase reporter cells according to Example <10-1>.

FIG. 42 shows induction of transactivation with iCP-SOX2 recombinant protein in luciferase reporter cells according to Example <10-2>.

FIG. 43 shows induction of transactivation with iCP-KLF4 recombinant protein in luciferase reporter cells according to Example <10-3>.

FIG. 44 shows induction of transactivation with iCP-CMYC recombinant protein in luciferase reporter cells according to Example <10-3>.

FIG. 45 shows induction of transactivation with iCP-NANOG recombinant protein in luciferase reporter cells according to Example <10-4>.

FIG. 46 shows induction of transactivation with iCP-LIN28 recombinant protein in luciferase reporter cells according to Example <10-5>.

FIG. 47 shows induction of formation of iPSC-like colonies with iCP-RFs recombinant protein: Protocol 1 according to Example <11-1>.

FIG. 48 shows induction of formation of iPSC-like colonies with iCP-RFs recombinant protein: Protocol 2 according to Example <11-2>.

FIG. 49 shows induction of formation of iPSC-like colonies with iCP-RFs recombinant protein: Protocol 3 according to Example <11-3>.

FIG. 50 shows induction of formation of iPSC-like colonies with iCP-RFs recombinant protein: Protocol 4 according to Example <11-4>.

FIG. 51 shows induction of formation of iPSC-like colonies with iCP-RFs recombinant protein: Protocol 5 according to Example <11-5>.

FIG. 52 shows expression of stem cell specific biomarkers of iPSC-like colonies according to Example 12.

MODE FOR INVENTION

1. Analysis of Reference Hydrophobic CPPs to Identify ‘Critical Factors’ for Development of Advanced MTDs

Previously reported MTDs were selected from a screen of more than 1,500 signal peptide sequences. Although the MTDs that have been developed did not have a common sequence or sequence motif, they were all derived from the hydrophobic (H) regions of signal sequences (HOURSS) that also lack common sequences or motifs except their hydrophobicity and the tendency to adopt alpha-helical conformations. The wide variation in H-region sequences may reflect prior evolution for proteins with membrane translocating activity and subsequent adaptation to the SRP/Sec61 machinery, which utilizes a methionine-rich signal peptide binding pocket in SRP to accommodate a wide-variety of signal peptide sequences.

Previously described hydrophobic CPPs (e.g. MTS/MTM and MTD) were derived from the hydrophobic regions present in the signal peptides of secreted and cell surface proteins. The prior art consists first, of ad hoc use of H-region sequences (MTS/MTM), and second, of H-region sequences (with and without modification) with highest CPP activity selected from a screen of 1,500 signal sequences (MTM). Second prior art, the modified H-region derived hydrophobic CPP sequences had advanced in diversity with multiple number of available sequences apart from MTS/MTM derived from fibroblast growth factor (FGF) 4. However, the number of MTDs that could be modified from naturally occurring secreted proteins are somewhat limited. Because there is no set of rules in determining their cell-permeability, no prediction for the cell-permeability of modified MTD sequences can be made before testing them.

The hydrophobic CPPs, like the signal peptides from which they originated, did not conform to a consensus sequence, and they had adverse effects on protein solubility when incorporated into protein cargo. We therefore set out to identify optimal sequence and structural determinants, namely critical factors (CFs), to design new hydrophobic CPPs with enhanced ability to deliver macromolecule cargoes including proteins into the cells and tissues while maintaining protein solubility. These newly developed CPPs, advanced macromolecule transduction domains (aMTDs) allowed almost infinite number of possible designs that could be designed and developed based on the critical factors. Also, their cell-permeability could be predicted by their character analysis before conducting any in vitro and/or in vivo experiments. These critical factors below have been developed by analyzing all published reference hydrophobic CPPs.

1-1. Analysis of Hydrophobic CPPs

Seventeen different hydrophobic CPPs (Table 1) published from 1995 to 2014 (Table 2) were selected. After physiological and chemical properties of selected hydrophobic CPPs were analyzed, 11 different characteristics that may be associated with cell-permeability have been chosen for further analysis. These 11 characteristics are as follows: sequence, amino acid length, molecular weight, pI value, bending potential, rigidity/flexibility, structural feature, hydropathy, residue structure, amino acid composition and secondary structure of the sequences (Table 3).

Table 1 shows the Summary of Published Hydrophobic Cell-Penetrating Peptides which were Chosen.

TABLE 1 # Pepides Origin Protein Ref.  1 MTM Homo sapiens NP_001998 1 Kaposi fibroblast growth factor (K-FGF)  2 MTS Homo sapiens NP_001998 Kaposi 2 fibroblastgrowth factor (K-FGF)  3 MTD10 Streptomyces coelicolor NP_625021 Glycosyl 8 hydrolase  4 MTD13 Streptarnyces coelicolor NP_639877 Putative 3 secreted protein  5 MTD47 Streptomyces coelicolor NP_627512 4 Secreted protein  6 MTD56 Homo sapiens P23274 Peptodyl-prolyl 5 cis-trans isomerase B precursor  7 MTD73 Drosophila AAA17887 Spatzle 5 melanogaster (spz) protein  8 MTD77 Homo sapiens NP_003231 6 Kaposi fibroblast growth factor (K-FGF)  9 MTD84 Phytophthora cactorum AAK63068 Phytotoxic 4 protein PcF precusor 10 MTD85 Streptomyces coelicolor NP_629842 7 Peptide transport system peptide binding protein 11 MTD86 Streptomyces coelicolor NP_99842 Peptide transport system secreted peptide binding protein 12 MTD103 Homo sapiens TMBV19 domain 8 Family member B 13 MTD132 Streptomyces coelicolor NP_628377 P60-family 4 secreted protein 14 MTD151 Streptomyces coelicolor NP_630126 Secreted a chitinase 15 MTD173 Streptomyces coelicolor NP_624384 4 Secreted protein 16 MTD174 Streptomyces coelicolor NP_733505 Large, 8 multifunctional secreted protein 17 MTD181 Neisseria CAB84257.1 Putative 4 meningitidis Z2491 secreted protein

Table 2 shows the Summarizes Reference Information.

TABLE 2 References # Title Journal Year Vol Issue Page 1 Inhibition of JOURNALOF 1995 270 24 14255 Nuclear Translocation BIOLOGICAL of Transcription CHEMISTRY Factor NF-kB by a Synthetic peptide Containing a Cell Membrane- permeable Motif and Nuclear Localization Sequence 2 Epigenetic NATURE 2001 19 10 929 Regulation of Gene BIOTECHNOLOGY Structure and Function with a Cell-Permeable Cre Recombinase 3 Cell-Permeable NM23 CANCER 2011 71 23 7216 Blocks the RESEARCH Maintenance and Progression of Established Pulmonary Metastasis 4 Antitumor Activity of MOLECULAR 2012 20 8 1540 Cell-Permeable THERAPY p18INK4c With Enhanced Membrane and Tissue Penetration 5 Antitumor Activity of CLINICAL 2012 19 3 680 Cell-Permeable CANCER RUNX3 Protein in RESEARCH Gastric Cancer Cells 6 The Effect of BIOMATERIALS 2013 34 26 6261 Intracellular Protein Delivery on the Anti- Tumor Activity of Recombinant Human Endostatin 7 Partial Somatic SCIENTIFIC 2014 4 10 4361 to Stem Cell REPORTS Transformations Induced By Cell-Permeable Reprogramming Factors 8 Cell-Permeable Parkin PLOS ONE 2014 9 7 17 Proteins Suppress Parkinson Disease-Associated Phenotypes in Cultured Cells and Animals

Table 3 shows the Characteristics of Published Hydrophobic Cell-Penetrating Peptides (A) which were Analyzed.

TABLE 3 Rigid- ity/ Struc- Flexi- tural bility Fea- Bend- (In- ture Hy- ing sta- (Ali- dro- Resi- A/a Secon- Molecu- Po- bility phatic pathy due Compo- dary Pep- Se- lar ten- Index: Index: (GRA- Struc- sition Struc- Car- # tides quence Length Weight pI tial II) AI) VY) ture A V L I P G ture go Ref.  1 MTM AAVALL 16 1,515.9 5.6 Bend- 45.5 220.0 2.4 Ali- 6 2 6 0 2 0 Helix p50 1 PAVLLA ing phatic LLAP Ring  2 MTS AAVLLP 12 1,147.4 5.6 Bend- 57.3 211.7 2.3 Ali- 4 2 4 0 2 0 No- CRE 2 VLLAAP ing phatic Helix Ring  3 MTD10 LGGAVV 16 1,333.5 5.5 Bend- 47.9 140.6 1.8 Ali- 7 4 1 0 2 2 Helix Par- 8 AAPVAA ing phatic kin AVAP Ring  4 MTD13 LAAAAL 11 1,022.3 5.5 Bend- 26.6 213.6 2.4 Ali- 5 1 4 0 1 0 No- RUNX 3 AVLPL ing phatic Helix 3 Ring  5 MTD47 AAAVPV 10   881.0 5.6 Bend- 47.5 176.0 2.4 Ali- 5 3 1 0 1 0 No- CMYC 4 LVAA ing phatic Helix Ring  6 MTD56 VLLAAA  9   854.1 5.5 No-  8.9 250.0 3.0 Ali- 4 1 3 1 0 0 Helix ES 5 LIA Bend- phatic ing Ring  7 MTD73 PVLLLL  7   737.9 6.0 No- 36.1 278.6 2.8 Ali- 1 1 4 0 1 0 Helix ES 5 A Bend- phatic ing Ring  8 MTD77 AVALLI  9   882.1 5.6 No- 30.3 271.1 3.3 Ali- 3 2 3 1 0 0 Helix NM23 6 LAV Bend- phatic ing Ring  9 MTD84 AVALVA 11   982.2 5.6 No-  9.1 212.7 3.1 Ali- 5 5 1 0 0 0 Helix OCT4 4 VVAVA Bend- phatic ing Ring 10 MTD85 LLAAAA 11 1,010.2 5.5 No-  9.1 231.8 2.7 Ali- 6 0 5 0 0 0 No- RUNX 7 ALLLA Bend- phatic Helix 3 ing Ring 11 MTD86 LLAAAA 11 1,010.2 5.5 No-  9.1 231.8 2.7 Ali- 6 0 5 0 0 0 No- SOX2 7 ALLLA Bend- phatic Helix ing Ring 12 MTD LALPVL  9   922.2 5.5 Bend- 51.7 271.1 2.8 Ali- 2 1 5 0 1 0 Helix p18 8 103 LLA ing phatic Ring 13 MTD AVVVPA 12 1,1194 5.6 Bend- 50.3 195.0 2.4 Ali- 4 4 1 1 2 0 No- LIN 4 132 LIVAAP ing phatic Helix 28 Ring 14 MTD AAAPVA  9 1,031.4 5.5 Bend- 73.1 120.0 1.6 Ali- No- Par- 8 151 AVP ing phatic Helix kin Ring 15 MTD AVIPIL  9   892.1 5.6 Bend- 48.5 216.7 2.4 Ali- 2 2 1 2 2 0 Helix KLF4 4 173 AVP ing phatic Ring 16 MTD LILLLP 12 1,011.8 5.5 Bend- 79.1 257.3 2.6 Ali- Helix Par- 8 174 AVALP ing phatic kin Ring 17 MTD AVLLLP  9   838.0 5.6 Bend- 51.7 206.7 2.4 Ali- 4 1 3 0 1 0 No- SOX2 4 181 AAA ing phatic Helix AVE 10.8 ± 1,011 ± 5.6 ± Pro- 40.1 ± 217.9 ± 2.5 ±  2.4   189.6 0.1 line 21.9  43.6 0.4 Pres- ence

Two peptide/protein analysis programs were used (ExPasy: SoSui: harrier.nagahama-i-bio.ac.jp) to determine various indexes and structural features of the peptide sequences and to design new sequence. Followings are important factors analyzed.

1-2. Characteristics of Analyzed Peptides: Length, Molecular Weight and pl Value

Average length, molecular weight and pl value of the peptides analyzed were 10.8±2.4, 1,011±189.6 and 5.6±0.1, respectively (Table 4)

Table 4 shows the Summarizes Critical Factors (CFs) of Published Hydrophobic Cell-Penetrating Peptides (A) which were Analyzed.

TABLE 4 Length: 10.8 ± 2.4 Molecular Weight: 1,011 ± 189.6 pl: 5.6 ± 0.1 Bending Potential (BP): Proline presences in the middle and/or the end of peptides, or No Proline. Instability Index (II): 40.1 ± 21.9 Residue Structure & Aliphatic Index (Al): 217.9 ± 43.6 Hydropathy (GRAVY): 2.5 ± 0.4 Aliphatic Ring: Non polar hydrophobic & aliphatic amino acid (A, V, L, I). Secondary Structure: α-Helix is favored but not required.

1-3. Characteristics of Analyzed Peptides: Bending Potential—Proline Position (PP)

Bending potential (bending or no-bending) was determined based on the fact whether proline (P) exists and/or where the amino acid(s) providing bending potential to the peptide in recombinant protein is/are located. Proline differs from the other common amino acids in that its side chain is bonded to the backbone nitrogen atom as well as the alpha-carbon atom. The resulting cyclic structure markedly influences protein architecture which is often found in the bends of folded peptide/protein chain.

Eleven out of 17 were determined as ‘Bending’ peptide which means that proline is present in the middle of sequence for peptide bending and/or located at the end of the peptide for protein bending. As indicated above, peptide sequences could penetrate the plasma membrane in a “bent” configuration. Therefore, bending or no-bending potential is considered as one of the critical factors for the improvement of current hydrophobic CPPs.

1-4. Characteristics of Analyzed Peptides: Rigidity/Flexibility—Instability Index (II)

Since one of the crucial structural features of any peptide is based on the fact whether the motif is rigid or flexible, which is an intact physicochemical characteristic of the peptide sequence, instability index (II) of the sequence was determined. The index value representing rigidity/flexibility of the peptide was extremely varied (8.9 to 79.1), but average value was 40.1±21.9 which suggested that the peptide should be somehow flexible, but not too much rigid or flexible (Table 3).

1-5. Characteristics of Analyzed Peptides: Structural Features—Structural Feature (Aliphatic Index: AI) and Hydropathy (Grand Average of Hydropathy: GRAVY)

Alanine (V), valine (V), leucine (L) and isoleucine (I) contain aliphatic side chain and are hydrophobic—that is, they have an aversion to water and like to cluster. These amino acids having hydrophobicity and aliphatic residue enable them to pack together to form compact structure with few holes. Analyzed peptide sequence showed that all composing amino acids were hydrophobic (A, V, L and I) except glycine (G) in only one out of 17 (MTD10—Table 3) and aliphatic (A, V, L, I, and P). Their hydropathic index (Grand Average of Hydropathy: GRAVY) and aliphatic index (AI) were 2.5±0.4 and 217.9±43.6, respectively. Their amino acid composition is also indicated in the Table 3.

1-6. Characteristics of Analyzed Peptides: Secondary Structure (Helicity)

As explained above, the CPP sequences may be supposed to penetrate the plasma membrane directly after inserting into the membranes in a “bent” configuration with hydrophobic sequences having α-helical conformation. In addition, our analysis strongly indicated that bending potential was crucial for membrane penetration. Therefore, structural analysis of the peptides was conducted to determine whether the sequences were to form helix or not. Nine peptides were helix and eight were not (Table 3). It seems to suggest that helix structure may not be required.

1-7. Determination of Critical Factors (CFs)

In the 11 characteristics analyzed, the following 6 are selected namely “Critical Factors” for the development of new hydrophobic CPPs—advanced MTDs: amino acid length, bending potential (proline presence and location), rigidity/flexibility (instability index: I), structural feature (aliphatic index: AI), hydropathy (GRAVY) and amino acid composition/residue structure (hydrophobic and aliphatic A/a) (Tables 3 and Table 4).

2. Analysis of Selected Hydrophobic CPPs to Optimize ‘Critical Factors’

Since the analyzed data of the 17 different hydrophobic CPPs (analysis A, Tables 3 and 4) previously developed during the past 2 decades showed high variation and were hard to make common- or consensus-features, analysis B (Tables 5 and 6) and C (Tables 7 and 8) were also conducted to optimize the critical factors for better design of improved CPPs-aMTDs. Therefore, 17 hydrophobic CPPs have been grouped into two groups and analyzed the groups for their characteristics in relation to the cell permeable property. The critical factors have been optimized by comparing and contrasting the analytical data of the groups and determining the common homologous features that may be critical for the cell permeable property.

2-1. Selective Analysis (B) of Peptides Used to Biologically Active Cargo Protein for In Vivo

In analysis B, eight CPPs were used with each biologically active cargo in vivo. Length was 11±3.2, but 3 out of 8 CPPs possessed little bending potential. Rigidity/Flexibility (instability index: II) was 41±15, but removing one [MTD85: rigid, with minimal II (9.1)] of the peptides increased the overall instability index to 45.6±9.3. This suggested that higher flexibility (40 or higher II) is potentially be better. All other characteristics of the 8 CPPs were similar to the analysis A, including structural feature and hydropathy (Tables 5 and 6).

Table 5 shows the Characteristics of Published Hydrophobic Cell-Penetrating Peptides (B): Selected CPPs That were Used to Each Cargo In Vivo.

TABLE 5 Rigid- ity/ Struc- Flexi- tural bility Fea- Bend- (In- ture Hy- ing sta- (Ali- dro- Resi- A/a Secon- Molecu- Po- bility phatic pathy due Compo- dary Pep- Se- lar ten- Index: Index: (GRA- Struc- sition Struc- Car- # tides quence Length Weight pI tial II) AI) VY) ture A V L I P G ture go Ref. 1 MTM AAVALL 16 1,515.9 5.6 Bend- 45.5 220.0 2.4 Ali- 6 2 6 0 2 0 Helix p50 1 PAVLLA ing phatic LLAP Ring 2 MTS AAVLLP 12 1,147.4 5.6 Bend- 57.3 211.7 2.3 Ali- 4 2 4 0 2 0 No- CRE 2 VLLAAP ing phatic Helix Ring 3 MTD10 LGGAVV 16 1,333.5 5.5 Bend- 47.9 140.6 1.8 Ali- 7 4 1 0 2 2 Helix Par- 8 AAPVAA ing phatic kin AVAP Ring 4 MTD73 PVLLLL  7   737.9 6.0 No- 36.1 278.6 2.8 Ali- 1 1 4 0 1 0 Helix ES 6 A Bend- phatic ing Ring 5 MTD77 AVALLI  9   882.1 5.6 No- 30.3 271.1 3.3 Ali- 3 2 3 1 0 0 Helix NM23 3 LAV Bend- phatic ing Ring 6 MTD85 LLAAAA 11 1,010.2 5.5 No-  9.1* 231.8 2.7 Ali- 6 0 5 0 0 0 No- RUNX 5 ALLLA Bend- phatic Helix 3 ing Ring 7 MTD LALPVL  9   922.2 5.5 Bend- 51.7 271.1 2.8 Ali- 2 1 5 0 1 0 Helix p18 4 103 LLA ing phatic Ring 8 MTD AVVVPA 12 1,119.4 5.6 Bend- 50.3 195.0 2.4 Ali- 4 4 1 1 2 0 No- LIN 7 132 IVLAAP ing phatic Helix 28 Ring AVE 11 ± 1,083 ± 5.6 ± Pro- 41 ± 227 ± 2.5 ±  3.2   252 0.1 line 15  47 0.4 Pres- ence *Removing the MTD85 increases II to 45.6 ± 9.3

Table 6 shows the Summarized Critical Factors of Published Hydrophobic Cell-Penetrating Peptides (B).

TABLE 6 Length: 11 ± 3.2 Molecular Weight: 1,083 ± 252 pl: 5.6 ± 0.1 Bending Potential (BP): Proline presences in the middle and/or the end of peptides, or No Proline. Instability Index (II): 41.0 ± 15 (* Removing the MTD85 increases II to 45.6 ± 9.3) Residue Structure & Aliphatic Index (Al): 227 ± 47 Hydropathy (GRAVY): 2.5 ± 0.4 Aliphatic Ring: Non-polar hydrophobic & aliphatic amino acid (A, V, L, I). Secondary Structure: α-Helix is favored but not required.

2-2. Selective Analysis (C) of Peptides that Provided Bending Potential and Higher Flexibility

To optimize the ‘Common Range and/or Consensus Feature of Critical Factor’ for the practical design of aMTDs and the random peptides (rPs or rPeptides), which were to prove that the ‘Critical Factors’ determined in the analysis A, B and C were correct to improve the current problems of hydrophobic CPPs—protein aggregation, low solubility/yield, and poor cell-/tissue-permeability of the recombinant proteins fused to the MTS/MTM or MTD, and non-common sequence and non-homologous structure of the peptides, empirically selected peptides were analyzed for their structural features and physicochemical factor indexes.

Hydrophobic CPPs which did noTo optimize the ‘Common Range and/or Consensus Feature of Critical Factor’ for the practical design of aMTDs and the random peptides (rPs or rPeptides), which were to prove that the ‘Critical Factors’ determined in the analysis A, B and C were correct to improve the current problems of hydrophobic CPPs—protein aggregation, low solubility/yield, and poor cell-/tissue-permeability of the recombinant proteins fused to the MTS/MTM or MTD, and non-common sequence and non-homologous structure of the peptides, empirically selected peptides were analyzed for their structural features and physicochemical factor indexes.

Hydrophobic CPPs which did not have a bending potential, rigid or too much flexible sequences (too much low or too much high Instability Index), or too low or too high hydrophobic CPPs were unselected, but secondary structure was not considered because helix structure of sequence was not required.

In analysis C, eight selected CPP sequences that could provide a bending potential and higher flexibility were finally analyzed (Table 7 and 8). Common amino acid length is 12 (11.6±3.0). Proline is presence in the middle of and/or the end of sequence. Rigidity/Flexibility (II) is 45.5 to 57.3 (Avg: 50.1±3.6). AI and GRAVY representing structural feature and hydrophobicity of the peptide are 204.7±37.5 and 2.4±0.3, respectively. All peptides are consisted with hydrophobic and aliphatic amino acids (A, V, L, I, and P). Therefore, analysis C was chosen as a standard for the new design of new hydrophobic CPPs—aMTDs.

Table 7 shows the Characteristics of Published Hydrophobic Cell-Penetrating Peptides (C): Selected CPPs that Provided Bending Potential and Higher Flexibility.

TABLE 7 Rigid- ity/ Struc- Flexi- tural bility Fea- Bend- (In- ture ing sta- (Ali- Hydro- Resi- A/a Secon- Molecu- Po- bility phatic pathy due Compo- dary Pep Se- lar ten- Index: Index: (GRA- Struc- sition Struc- Car- # tides quence Length Weight pI tial II) AI) VY) ture A V L I P G ture go Ref. 1 MTM AAVALL 16 1515.9 5.6 Bend- 45.5 220.0 2.4 Ali- 6 2 6 0 2 0 Helix p50 1 PAVLLA ing phatic LLAP Ring 2 MTS AAVLLP 12 1147.4 5.6 Bend- 57.3 211.7 2.3 Ali- 4 2 4 0 2 0 No- CRE 2 VLLAAP ing phatic Helix Ring 3 MTD10 LGGAVV 16 1333.5 5.5 Bend- 47.9 140.6 1.8 Ali- 7 4 1 0 2 2 Helix Par- 8 AAPVAA ing phatic kin AVAP Ring 4 MTD47 AAAVPV 10  881.0 5.6 Bend- 47.5 176.0 2.4 Ali- 5 3 1 0 1 0 No- CMYC 4 LVAA ing phatic Helix Ring 5 MTD LALPVL  9  922.2 5.5 Bend- 51.7 271.1 2.8 Ali- 2 1 5 0 1 0 Helix p18 8 103 LLA ing phatic Ring 6 MTD AVVVPA 12 1119.4 5.6 Bend- 50.3 195.0 2.4 Ali- 4 4 1 1 2 0 No- LIN 4 132 IVLAAP ing phatic Helix 28 Ring 7 MTD AVIPIL  9  892.1 5.6 Bend- 48.5 216.7 2.4 Ali- 2 2 1 2 2 0 Helix KLF4 4 173 AVP ing phatic Ring 8 MTD AVLLLP  9  838.0 5.6 Bend- 51.7 206.7 2.4 Ali- 4 1 3 0 1 0 No- SOX2 4 181 AAA ing phatic Helix Ring AVE 11.6 ± 1081.2 ± 5.6 ± Pro- 50.1 ± 204.7 ± 2.4 ±  3.0  244.6 0.1 line  3.6  37.5 0.3 Pres- ence

Table 8 shows the Summarized Critical Factors of Published Hydrophobic Cell-Penetrating Peptides (C).

TABLE 8 Length: 11.6 ± 3.0 Molecular Weight: 1,081.2 ± 224.6 pl: 5.6 t 0.1 Bending Potential (BP): Proline presences in the middle and/or the end of peptides. Instability index (II): 50.1 ± 3.6 Residue Structure & Aliphatic Index (Al): 204.7 ± 37.5 Hydropathy (GRAVY): 2.4 ± 0.3 Aliphatic Ring: Non-polar hydrophobic & aliphatic amino acid (A, V, L, I). Secondary Structure: α-Helix is favored but not required. 3. New Design of Improved Hydrophobic CPPs—aMTDs Based on the Optimized Critical Factors

3-1. Determination of Common Sequence and/or Common Homologous Structure

As mentioned above, H-regions of signal sequence (HOURSS)-derived CPPs (MTS/MTM and MTD) do not have a common sequence, sequence motif, and/or common-structural homologous feature. According to one embodiment of the present invention, the aim is to develop improved hydrophobic CPPs formatted in the common sequence- and structural-motif which satisfy newly determined ‘Critical Factors’ to have ‘Common Function,’ namely, to facilitate protein translocation across the membrane with similar mechanism to the analyzed reference CPPs. Based on the analysis A, B and C, the common homologous features have been analyzed to determine the critical factors that influence the cell-permeability. The range value of each critical factor has been determined to include the analyzed index of each critical factor from analysis A, B and C to design novel aMTDs (Table 9). These features have been confirmed experimentally with newly designed aMTDs in their cell-permeability.

Table 9 shows the Comparison The Range/Feature of Each Critical Factor Between The Value of Analyzed CPPs and The Value Determined for New Design of Novel aMTDs Sequences.

TABLE 9 Summarized Critical Factors of aMTD Selected CPPs Newly Designed CPPs Critical Factor Range Range Bending Proline presences Proline presences in the Potential in the middle and/or middle (5′, 6′, 7′ or 8′ and (Proline Position: PP) at the end of peptides at the end of peptides Rigidity/Flexibility 45.5-57.3 (50.1 ± 3.6) 40-60 (Instability Index: II) Structural Feature 140.6-220.0 180-220 (Aliphatic Index: Al) (204.7 ± 37.5) Hydropathy 1.8-2.8 (2.4 ± 0.3) 2.1-2.6 (Grand Average of Hydropathy GRAVY) Length 11.6 ± 3.0  9-13 (Number of Amino Acid) Amino acid A, V, I, L, P A, V, I, L, P Composition

In Table 9, universal common features and sequence/structural motif are provided. Length is 9 to 13 amino acids, and bending potential is provided with the presence of proline in the middle of sequence (at 5′, 6′, 7′ or 8′ amino acid) for peptide bending and at the end of peptide for recombinant protein bending and Rigidity/Flexibility of aMTDs is II >40 are described in Table 9.

3-2. Critical Factors for Development of Advanced MTDs

Recombinant cell-permeable proteins fused to the hydrophobic CPPs to deliver therapeutically active cargo molecules including proteins into live cells had previously been reported, but the fusion proteins expressed in bacteria system were hard to be purified as a soluble form due to their low solubility and yield. To address the crucial weakness for further clinical development of the cell-permeable proteins as protein-based biotherapeutics, greatly improved form of the hydrophobic CPP, named as advanced MTD (aMTD) has newly been developed through critical factors-based peptide analysis. The critical factors used for the current invention of the aMTDs are herein (Table 9).

1. Amino Acid Length: 9 to 13

2. Bending Potential (Proline Position: PP)

: Proline presences in the middle (from 5′ to 8′ amino acid) and at the end of sequence

3. Rigidity/Flexibility (Instability Index: II): 40 to 60

4. Structural Feature (Aliphatic Index: AI): 180 to 220

5. Hydropathy (GRAVY): 2.1 to 2.6

6. Amino Acid Composition: Hydrophobic and Aliphatic amino acids to A, V, L, I and P

3-3. Design of Potentially Best aMTDs that all Critical Factors are Considered and Satisfied

After careful consideration of six critical factors derived from analysis of unique features of hydrophobic CPPs, advanced macromolecule transduction domains (aMTDs) have been designed and developed based on the common 12 amino acid platform which satisfies the critical factors including amino acid length (9 to 13) determined from the analysis. [General formula] X1-X2-X3-X4-U5-U6-U7-U8-X9-X10-X11-P

Unlike previously published hydrophobic CPPs that require numerous experiments to determine their cell-permeability, newly developed aMTD sequences could be designed by performing just few steps as follows using above mentioned platform to follow the determined range value/feature of each critical factor.

First, prepare the 12 amino acid sequence platform for aMTD. Second, place proline (P) in the end (12′) of sequence and determine where to place proline in one of four U(s) in 5′, 6′, 7′, and 8. Third, alanine (A), valine (V), leucine (L) or isoleucine (I) is placed in either X(s) and/or U(s), where proline is not placed. Lastly, determine whether the amino acid sequences designed based on the platform, satisfy the value or feature of six critical factors to assure the cell permeable property of aMTD sequences. Through these processes, numerous novel aMTD sequences have been constructed. The expression vectors for preparing non-functional cargo recombinant proteins fused to each aMTD, expression vectors have been constructed and forcedly expressed in bacterial cells. These aMTD-fused recombinant proteins have been purified in soluble form and determined their cell-permeability quantitatively. aMTD sequences have been newly designed, numbered from 1 to 240, as shown in Tables 10 to 15. In Tables 10 to 15, sequence ID Number is a sequence listings for reference, and aMTD numbers refer to amino acid listing numbers that actually have been used at the experiments. For further experiments, aMTD numbers have been used. In addition, polynucleotide sequences shown in the sequence lists have been numbered from SEQ ID NO: 241 to SEQ ID NO: 480.

Tables 10 to 15 show the 240 new hydrophobic aMTD sequences that were developed to satisfy all critical factors.

TABLE 10 Rigidity/ Sturctural Sequence Flexibility Feature Hydropathy Residue ID Number aMTD Sequences Length (II) (AI) (GRAVY) Structure 1 1 AAALAPVVLALP 12 57.3 187.5 2.1 Aliphatic 2 2 AAAVPLLAVVVP 12 41.3 195.0 2.4 Aliphatic 3 3 AALLVPAAVLAP 12 57.3 187.5 2.1 Aliphatic 4 4 ALALLPVAALAP 12 57.3 195.8 2.1 Aliphatic 5 5 AAALLPVALVAP 12 57.3 187.5 2.1 Aliphatic 6 11 VVALAPALAALP 12 57.3 187.5 2.1 Aliphatic 7 12 LLAAVPAVLLAP 12 57.3 211.7 2.3 Aliphatic 8 13 AAALVPVVALLP 12 57.3 203.3 2.3 Aliphatic 9 21 AVALLPALLAVP 12 57.3 211.7 2.3 Aliphatic 10 22 AVVLVPVLAAAP 12 57.3 195.0 2.4 Aliphatic 11 23 VVLVLPAAAAVP 12 57.3 195.0 2.4 Aliphatic 12 24 IALAAPALIVAP 12 50.2 195.8 2.2 Aliphatic 13 25 IVAVAPALVALP 12 50.2 203.3 2.4 Aliphatic 14 42 VAALPVVAVVAP 12 57.3 186.7 2.4 Aliphatic 15 43 LLAAPLVVAAVP 12 41.3 187.5 2.1 Aliphatic 16 44 ALAVPVALLVAP 12 57.3 203.3 2.3 Aliphatic 17 61 VAALPVLLAALP 12 57.3 211.7 2.3 Aliphatic 18 62 VALLAPVALAVP 12 57.3 203.3 2.3 Aliphatic 19 63 AALLVPALVAVP 12 57.3 203.3 2.3 Aliphatic

TABLE 11 Rigidity/ Sturctural Sequence Flexibility Feature Hydropathy Residue ID Number aMTD Sequences Length (II) (AI) (GRAVY) Structure 20 64 AIVALPVAVLAP 12 50.2 203.3 2.4 Aliphatic 21 65 IAIVAPVVALAP 12 50.2 203.2 2.4 Aliphatic 22 81 AALLPALAALLP 12 57.2 204.2 2.1 Aliphatic 23 82 AVVLAPVAAVLP 12 57.3 195.0 2.4 Aliphatic 24 83 LAVAAPLALALP 12 41.2 195.8 2.1 Aliphatic 25 84 AAVAAPLLLALP 12 41.3 195.2 2.1 Aliphatic 26 85 LLVLPAAALAAP 12 57.3 195.2 2.1 Aliphatic 27 101 LVALAPVAAVLP 12 57.2 203.3 2.3 Aliphatic 20 102 LALAPAALALLP 12 57.2 204.2 2.1 Aliphatic 29 103 ALIAAPILALAP 12 57.2 204.2 2.2 Aliphatic 30 104 AVVAAPLVLALP 12 41.3 203.3 2.3 Aliphatic 31 105 LLALAPAALLAP 12 57.3 204.1 2.1 Aliphatic 32 121 AIVALPALALAP 12 50.2 195.8 2.2 Aliphatic 33 123 AAIIVPAALLAP 12 50.2 195.8 2.2 Aliphatic 34 124 IAVALPALIAAP 12 50.3 195.2 2.2 Aliphatic 35 141 AVIVLPALAVAP 12 50.2 203.3 2.4 Aliphatic 36 143 AVLAVPAVLVAP 12 57.3 195.0 2.4 Aliphatic 37 144 VLAIVPAVALAP 12 50.2 203.3 2.4 Aliphatic 38 14$ LLAVVPAVALAP 12 57.2 203.3 2.3 Aliphatic 39 161 AVIALPALIAAP 12 57.3 195.2 2.2 Aliphatic 40 162 AVVALPAALIVP 12 50.2 203.2 2.4 Aliphatic 41 163 LALVLPAALAAP 12 57.3 195.2 2.1 Aliphatic 42 164 LAAVLPALLAAP 12 57.3 195.2 2.1 Aliphatic 43 165 ALAVPVALAIVP 12 50.2 203.3 2.4 Aliphatic 44 182 ALIAPVVALVAP 12 57.2 203.3 2.4 Aliphatic 45 183 LLAAPVVIALAP 12 57.3 211.6 2.4 Aliphatic 46 184 LAAIVPAIIAVP 12 50.2 211.6 2.4 Aliphatic 47 185 AALVLPLIIAAP 12 41.3 220.0 2.4 Aliphatic 48 201 LALAVPALAALP 12 57.2 195.2 2.1 Aliphatic 49 204 LIAALPAVAALP 12 57.2 195.2 2.2 Aliphatic 50 205 ALALVPAIAALP 12 57.2 195.2 2.2 Aliphatic 51 221 AAILAPIVALAP 12 50.2 195.2 2.2 Aliphatic 52 222 ALLIAPAAVIAP 12 57.2 195.2 2.2 Aliphatic 53 223 AILAVPIAVVAP 12 57.3 203.2 2.4 Aliphatic 54 224 ILAAVPIALAAP 12 57.2 195.2 2.2 Aliphatic 55 225 VAALLPAAAVLP 12 57.3 187.5 2.1 Aliphatic 56 241 AAAVVPVLLVAP 12 57.3 195.0 2.4 Aliphatic 57 242 AALLVPALVAAP 12 57.2 187.5 2.1 Aliphatic 58 243 AAVLLPVALAAP 12 57.3 187.5 2.1 Aliphatic 59 245 AAALAPVLALVP 12 57.2 187.5 2.1 Aliphatic 60 261 LVLVPLLAAAAP 12 41.3 211.6 2.3 Aliphatic 61 262 ALIAVPAIIVAP 12 50.2 211.6 2.4 Aliphatic 62 263 ALAVIPAAAILP 12 54.2 195.2 2.2 Aliphatic 63 264 LAAAPVVIVIAP 12 50.2 203.3 2.4 Aliphatic 64 265 VLAIAPLLAAVP 12 41.3 211.6 2.2 Aliphatic 65 281 ALIVLPAAVAVP 12 50.2 203.2 2.4 Aliphatic 66 282 VLAVAPALIVAP 12 50.2 203.3 2.4 Aliphatic 67 283 AALLAPALIVAP 12 50.2 195.2 2.2 Aliphatic 68 284 ALIAPAVALIVP 12 50.2 211.7 2.4 Aliphatic 69 285 AIVLLPAAVVAP 12 50.2 203.3 2.4 Aliphatic

TABLE 12 Rigidity/ Sturctural Sequence Flexibility Feature Hydropathy Residue ID Number aMTD Sequences Length (II) (AI) (GRAVY) Structure 70 301 VIAAPVLAVLAP 12 57.3 203.3 2.4 Aliphatic 71 302 LALAPALALLAP 12 57.3 204.2 2.1 Aliphatic 72 304 AIILAPIAAIAP 12 57.3 204.2 2.3 Aliphatic 73 305 IALAAPILLAAP 12 57.3 204.2 2.2 Aliphatic 74 321 IVAVALPALAVP 12 50.2 203.3 2.2 Aliphatic 75 322 VVAIVLPALAAP 12 50.2 203.3 2.3 Aliphatic 76 323 IVAVALPVALAP 12 50.2 203.3 2.2 Aliphatic 77 324 IVAVALPAALVP 12 50.2 203.3 2.2 Aliphatic 78 325 IVAVALPAVALP 12 50.2 203.3 2.3 Aliphatic 79 341 IVAVALPAVLAP 12 50.2 203.3 2.3 Aliphatic 80 342 VIVALAPAVLAP 12 50.2 203.3 2.2 Aliphatic 81 343 IVAVALPALVAP 12 50.2 203.3 2.2 Aliphatic 82 345 ALLIVAPVAVAP 12 50.2 203.3 2.3 Aliphatic 83 361 AVVIVAPAVIAP 12 50.2 195.0 2.4 Aliphatic 84 363 AVLAVAPALIVP 12 50.2 203.3 2.3 Aliphatic 85 364 LVAAVAPALIVP 12 50.2 203.3 2.3 Aliphatic 86 365 AVIVVAPALLAP 12 50.2 203.3 2.3 Aliphatic 87 381 VVAIVLPAVAAP 12 50.2 195.0 2.4 Aliphatic 88 382 AAALVIPAILAP 12 54.9 195.8 2.2 Aliphatic 89 383 VIVALAPALLAP 12 50.2 211.6 2.3 Aliphatic 90 384 VIVAIAPALLAP 12 50.2 211.6 2.4 Aliphatic 91 385 IVAIAVPALVAP 12 50.2 203.3 2.4 Aliphatuc 92 401 AALAVIPAAILP 12 54,9 195.8 2.2 Aliphatic 93 402 ALAAVIPAAILP 12 54.9 196.2 2.2 Aliphatic 94 403 AAALVIPAAILP 12 54.9 195.8 2.2 Aliphatic 95 404 LAAAVIPAAILP 12 54.9 195.2 2.2 Aliphatic 96 405 LAAAVIPVAILP 12 54.9 211.7 2.4 Aliphatic 97 421 AAILAAPLIAVP 12 57.3 195.8 2.2 Aliphatic 98 422 VVAILAPLLAAP 12 57.3 211.7 2.4 Aliphatic 99 424 AVVVAAPVLALP 12 57.3 195.0 2.4 Aliphatic 100 425 AVVAIAPVLALP 12 57.3 200.3 2.4 Aliphatic 101 442 ALAALVPAVLVP 12 57.3 203.3 2.3 Aliphatic 102 443 ALAALVPVALVP 12 57.3 203.3 2.3 Aliphatic 103 444 LAAALVPVALVP 12 57.3 203.3 2.2 Aliphatic 104 445 ALAALVPALVVP 12 57.3 203.3 2.3 Aliphatic 105 461 IAAVIVPAVALP 12 60.2 203.3 2.4 Aliphatic 106 482 IAAVLVPAVALP 12 57.3 203.3 2.4 Aliphatic 107 463 AVAILVPLLAAP 12 57.3 211.7 2.4 Aliphatic 108 464 AVVILVPLAAAP 12 57.3 203.3 2.4 Aliphatic 109 485 IAAVIVPVAALP 12 50.2 203.3 2.4 Aliphatic 110 481 AIAIAIVPVALP 12 50.2 211.6 2.4 Aliphatic 111 482 ILAVAAIPVAVP 12 54.9 203.3 2.4 Aliphatic 112 483 ILAAAIIPAALP 12 54.9 204.1 2.2 Aliphatic 113 484 LAVVLAAPAIVP 12 50.2 203.3 2.4 Aliphatic 114 485 AILAAIVPLAVP 12 50.2 211.2 2.4 Aliphatic 115 501 VIVALAVPALAP 12 50.2 203.3 2.4 Aliphatic 116 502 AIVALAVPVLAP 12 50.2 203.3 2.4 Aliphatic 117 503 AAIIIVLPAALP 12 50.2 220.0 2.4 Aliphatic 118 504 LIVALAVPALAP 12 50.2 211.7 2.4 Aliphatic 119 505 AIIIVIAPAAAP 12 50.2 195.8 2.3 Aliphatic

TABLE 13 Rigidity/ Sturctural Sequence Flexibility Feature Hydropathy Residue ID Number aMTD Sequences Length (II) (AI) (GRAVY) Structure 120 521 LAALIVVPAVAP 12 60.2 203.3 2.4 Aliphatic 121 522 ALLVIAVPAVAP 12 57.3 203.3 2.4 Aliphatic 122 524 AVALIVVPALAP 12 50.2 203.3 2.4 Aliphatic 123 625 ALAIVVAPVAVP 12 50.2 195.0 2.4 Aliphatic 124 541 LLALIIAPAAAP 12 57.3 204.1 2.1 Aliphatic 125 542 ALALIIVPAVAP 12 50.2 211.6 2.4 Aliphatic 126 543 LLAALIAPAALP 12 57.3 204.1 2.1 Aliphatic 127 544 IVALIVAPAAVP 12 43.1 203.3 2.4 Aliphatic 128 645 VVLVLAAPAAVP 12 57.3 195.0 2.3 Aliphatic 129 561 AAVAIVLPAVVP 12 50.2 195.0 2.4 Aliphatic 130 562 ALIAAIVPALVP 12 50.2 211.7 2.4 Aliphatic 131 563 ALAVIVVPALAP 12 50.2 203.3 2.4 Aliphatic 132 664 VAIALIVPALAP 12 60.2 211.7 2.4 Aliphatic 133 565 VAIVLVAPAVAP 12 50.2 195.2 2.4 Aliphatic 134 582 VAVALIVPALAP 12 50.2 203.3 2.4 Aliphatic 135 583 AVILALAPIVAP 12 50.2 211.6 2.4 Aliphatic 136 585 ALIVAIAPALVP 12 50.2 211.6 2.4 Aliphatic 137 601 AAILIAVPIAAP 12 57.3 195.2 2.3 Aliphatic 138 602 VIVALAAPVLAP 12 50.2 203.3 2.4 Aliphatic 139 603 VLVALAAPVIAP 12 57.3 203.3 2.4 Aliphatic 140 604 VALIAVAPAVVP 12 57.3 195.0 2.4 Aliphatic 141 605 VIAAVLAPVAVP 12 57.3 195.0 2.4 Aliphatic 142 622 ALIVLAAPVAVP 12 50.2 203.3 2.4 Aliphatic 143 623 VAAAIALPAIVP 12 50.2 187.5 2.3 Aliphatic 144 625 ILAAAAAPLIVP 12 50.2 195.8 2.2 Aliphatic 145 643 LALVLAAPAIVP 12 50.2 211.6 2.4 Aliphatic 146 645 ALAVVALPAIVP 12 50.2 203.3 2.4 Aliphatic 147 661 AAILAPIVAALP 12 50.2 195.8 2.2 Aliphatic 148 664 ILIAIAIPAAAP 12 54.9 204.1 2.3 Aliphatic 149 665 LAIVLAAPVAVP 12 50.2 203.3 2.3 Aliphatic 150 666 AAIAIIAPAIVP 12 50.2 195.2 2.3 Aliphatic 151 667 LAVAIVAPALVP 12 50.2 203.3 2.3 Aliphatic 152 683 LAIVLAAPAVLP 12 50.2 211.7 2.4 Aliphatic 153 684 AAIVLALPAVLP 12 50.2 211.7 2.4 Aliphatic 154 685 ALLVAVLPAALP 12 57.3 211.7 2.3 Aliphatic 155 686 AALVAVLPVALP 12 57.3 203.3 2.3 Aliphatic 156 687 AILAVALPLLAP 12 57.3 220.0 2.3 Aliphatic 157 703 IVAVALVPALAP 12 50.2 203.3 2.4 Aliphatic 158 705 IVAVALLPALAP 12 50.2 211.7 2.4 Aliphatic 159 706 IVAVALLPAVAP 12 50.2 203.3 2.4 Aliphatic 160 707 IVALAVLPAVAP 12 50.2 203.3 2.4 Aliphatic 161 724 VAVLAVLPALAP 12 57.3 203.3 2.3 Aliphatic 162 725 IAVLAVAPAVLP 12 57.3 203.3 2.3 Aliphatic 163 725 LAVAIIAPAVAP 12 57.3 187.5 2.2 Aliphatic 164 727 VALAIALPAVLP 12 57.3 211.6 2.3 Aliphatic 165 743 AIAIALVPVALP 12 57.3 211.6 2.4 Aliphatic 166 744 AAVVIVAPVALP 12 50.2 195.0 2.4 Aliphatic 167 746 VAIIVVAPALAP 12 50.2 203.3 2.4 Aliphatic 168 747 VALLAIAPALAP 12 57.3 195.8 2.2 Aliphatic 169 763 VAVLIAVPALAP 12 67.3 203.3 2.3 Aliphatic

TABLE 14 Rigidity/ Sturctural Sequence Flexibility Feature Hydropathy Residue ID Number aMTD Sequences Length (II) (AI) (GRAVY) Structure 170 764 AVALAVLPAVVP 12 57.3 196.0 2.3 Aliphatic 171 765 AVALAVVPAVLP 12 57.3 196.0 2.3 Aliphatic 172 766 IVVIAVAPAVAP 12 50.2 195.0 2.4 Aliphatic 173 767 IVVAAVVPALAP 12 50.2 195.0 2.4 Aliphatic 174 783 IVALVPAVAIAP 12 50.2 203.3 2.5 Aliphatic 175 784 VAALPAVALVVP 12 57.3 195.0 2.4 Aliphatic 176 786 LVAIAPLAVLAP 12 41.3 211.7 2.4 Aliphatic 177 787 AVALVPVIVAAP 12 50.2 195.0 2.4 Aliphatic 178 788 AIAVAIAPVALP 12 57.3 187.5 2.3 Aliphatic 179 803 AIALAVPVLALP 12 57.3 211.7 2.4 Aliphatic 180 805 LVLIAAAPIALP 12 41.3 220.0 2.4 Aliphatic 181 806 LVALAVPAAVLP 12 57.3 203.3 2.3 Aliphatic 182 807 AVALAVPALVLP 12 57.3 203.3 2.3 Aliphatic 183 808 LVVLAAAPLAVP 12 41.3 203.3 2.3 Aliphatic 184 809 LIVLAAPALAAP 12 50.2 195.3 2.2 Aliphatic 185 810 VIVLAAPALAAP 12 50.2 187.5 2.2 Aliphatic 186 811 AVVLAVPALAVP 12 57.3 195.0 2.3 Aliphatic 187 824 LIIVAAAPAVAP 12 50.2 187.5 2.3 Aliphatic 188 825 IVAVIVAPAVAP 12 43.2 195.0 2.5 Aliphatic 189 826 LVALAAPIIAVP 12 41.3 211.7 2.4 Aliphatic 190 827 IAAVLAAPALVP 12 57.3 187.5 2.2 Aliphatic 191 828 IALLAAPIIAVP 12 41.3 220.0 2.4 Aliphatic 192 828 AALALVAPVIVP 12 50.2 203.3 2.4 Aliphatic 193 830 IALVAAPVALVP 12 57.3 203.3 2.4 Aliphatic 194 831 IIVAVAPAAIVP 12 43.2 203.3 2.5 Aliphatic 195 832 AVAAIVPVIVAP 12 43.2 195.0 2.5 Aliphatic 196 843 AVLVLVAPAAAP 12 41.3 219.2 2.5 Aliphatic 197 844 VVALLAPLIAAP 12 41.3 211.8 2.4 Aliphatic 198 845 AAVVIAPLLAVP 12 41.3 203.3 2.4 Aliphatic 199 846 IAVAVAAPLLVP 12 41.3 203.3 2.4 Aliphatic 200 847 LVAIVVLPAVAP 12 50.2 219.2 2.6 Aliphatic 201 848 AVAIVVLPAVAP 12 50.2 195.0 2.4 Aliphatic 202 849 AVILLAPLIAAP 12 57.3 220.0 2.4 Aliphatic 203 850 LVIALAAPVALP 12 57.3 211.7 2.4 Aliphatic 204 851 VLAVVLPAVALP 12 57.3 219.2 2.5 Aliphatic 205 852 VLAVAAPAVLLP 12 57.3 203.3 2.3 Aliphatic 206 863 AAVVLLPIIAAP 12 41.3 211.7 2.4 Aliphatic 207 864 ALLVIAPAIAVP 12 57.3 211.7 2.4 Aliphatic 208 865 AVLVIAVPAIAP 12 57.3 203.3 2.5 Aliphatic 209 867 ALLVVIAPLAAP 12 41.3 211.7 2.4 Aliphatic 210 868 VLVAAILPAAIP 12 54.9 211.7 2.4 Aliphatic 211 870 VLVAAVLPIAAP 12 41.3 203.3 2.4 Aliphatic 212 872 VLAAAVLPLVVP 12 41.3 219.2 2.5 Aliphatic 213 875 AIAIVVPAVAVP 12 50.2 196.0 2.4 Aliphatic 214 877 VAIIAVPAVVAP 12 57.3 195.0 2.4 Aliphatic 215 878 IVALVAPAAVVP 12 50.2 196.0 2.4 Aliphatic 216 879 AAIVLLPAVVVP 12 50.2 219.1 2.5 Aliphatic 217 881 AALIVVPAVAVP 12 50.2 195.0 2.4 Aliphatic 218 882 AIALVVPAVAVP 12 57.3 196.0 2.4 Aliphatic 219 883 LAIVPAAIAALP 12 50.2 195.8 2.2 Aliphatic

TABLE 15 Rigidity/ Sturctural Sequence Flexibility Feature Hydropathy Residue ID Number aMTD Sequences Length (II) (AI) (GRAVY) Structure 220 885 LVAIAPAVAVLP 12 57.3 203.3 2.4 Aliphatic 221 887 VLAVAPAVAVLP 12 57.3 185.0 2.4 Aliphatic 222 888 ILAVVAIPAAAP 12 54.9 187.5 2.3 Aliphatic 223 889 ILVAAAPIAALP 12 57.3 195.8 2.2 Aliphatic 224 891 ILAVAAIPAALP 12 54.9 195.8 2.2 Aliphatic 225 893 VIAIPAILAAAP 12 54.9 195.8 2.3 Aliphatic 226 895 AIIIVVPAIAAP 12 50.2 211.3 2.5 Aliphatic 227 896 AILIVVAPIAAP 12 50.2 211.7 2.5 Aliphatic 228 897 AVIVPVAIIAAP 12 50.2 203.3 2.5 Aliphatic 229 899 AVVIALPAVVAP 12 57.3 195.0 2.4 Aliphatic 230 900 ALVAVIAPVVAP 12 57.3 195.0 2.4 Aliphatic 231 901 ALVAVLPAVAVP 12 57.3 195.0 2.4 Aliphatic 232 902 ALVAPLLAVAVP 12 41.3 203.3 2.3 Aliphatic 233 904 AVLAVVAPVVAP 12 57.3 186.7 2.4 Aliphatic 234 905 AVIAVAPLVVAP 12 41.3 195.0 2.4 Aliphatic 235 906 AVIALAPVVVAP 12 57.3 195.0 2.4 Aliphatic 236 907 VAIALAPVVVAP 12 57.3 195.0 2.4 Aliphatic 237 908 VALALAPVVVAP 12 57.3 195.0 2.3 Aliphatic 238 910 VAALLPAVVVAP 12 57.3 195.0 2.3 Aliphatic 239 911 VALALPAVVVAP 12 57.3 195.0 2.3 Aliphatic 240 912 VALLAPAVVVAP 12 57.3 195.0 2.3 Aliphatic 52.6 ± 5.1 201.7 ± 7.8 2.3 ± 0.1

3-4. Design of the Peptides that Did not Satisfy at Least One Critical Factor

To demonstrate that one embodiment of the present invention of new hydrophobic CPPs—aMTDs, which satisfy all critical factors described above, are correct and rationally designed, the peptides which do not satisfy at least one critical factor have also been designed. Total of 31 rPeptides (rPs) are designed, developed and categorized as follows: no bending peptides, either no proline in the middle as well at the end and/or no central proline; rigid peptides (II<40); too much flexible peptides; aromatic peptides (aromatic ring presences); hydrophobic, with non-aromatic peptides but have amino acids other than A, V, L, I, P or additional proline residues; hydrophilic, but non-aliphatic peptides.

3-4-1. Peptides that do not Satisfy the Bending Potential

Table 16 shows the peptides that do not have any proline in the middle (at 5′, 6′, 7′ or 8′) and at the end of the sequences. In addition, Table 16 describes the peptides that do not have proline in the middle of the sequences. All these peptides are supposed to have no-bending potential.

TABLE 16 Proline Rigidity/ Sturctural Position Flexibility Feature Hydropathy Group rPeptide ID Sequences Length (PP) (II) (AI) (GRAVY) No Bending Peptides 931 AVLIAPAILAAA 12  6 57.3 204.2 2.5 (No Praline at 5, 6, 7 936 ALLILAAAVAAP 12 12 41.3 204.2 2.4 or 8 and/or 12) 152 LAAAVAAVAALL 12 None 9.2 204.2 2.7 27 LAIVAAAAALVA 12 None 2.1 204.2 2.8 935 ALLILPAAAVAA 12  6 57.3 204.2 2.4 670 ALLILAAAVAAL 12 None 25.2 236.6 2.3 934 LILAPAAVVAAA 12  5 57.3 195.8 2.5 37 TTCSQQQVCTNG 12 None 53.1 0.0 −1.1 16 NNSCTTYTNGSQ 12 None 47.4 0.0 −1.4 113 PVAVALLIAVPP 12  1, 11, 12 57.3 195.0 2.1

3-4-2. Peptides that do not Satisfy the Rigidity/Flexibility

To prove that rigidity/flexibility of the sequence is a crucial critical factor, rigid (Avg. II: 21.8±6.6) and too high flexible sequences (Avg. II: 82.3±21.0) were also designed. Rigid peptides that instability index is much lower than that of new aMTDs (II: 41.3 to 57.3, Avg. II: 53.3±5.7) are shown in Table 17. Bending, but too high flexible peptides that II is much higher than that of new aMTDs are also provided in Table 18.

TABLE 17 Proline Rigidity/ Sturctual Length Position Flexibility Feature Hydropathy Group rPeptide ID Sequences (PP) (II) (AI) (GRAVY) Rigid Peptides 226 ALVAAIPALAIP 12 6 20.4 195.8 2.2 (II < 50) 6 VIAMIPAAFWVA 12 6 15.7 146.7 2.2 750 LAIAAIAPLAIP 12 8, 12 22.8 204.2 2.2 26 AAIALAAPLAIV 12 8 18.1 204.2 2.5 527 LVLAAVAPIAIP 12 8, 12 22.8 211.7 2.4 466 IIAAAAPLAIIP 12 7, 12 22.8 204.2 2.3 167 VAIAIPAALAIP 12 6, 12 20.4 195.8 2.3 246 VVAVPLLVAFAA 12 5 25.2 195.0 2.7 426 AAALAIPLAIIP 12 7, 12 4.37 204.2 2.2 606 AAAIAAIPIIIP 12 8, 12 4.4 204.2 2.4 66 AGVLGGPIMGVP 12 7, 12 35.5 121.7 1.3 248 VAAIVPIAALVP 12 6, 12 34.2 203.3 2.5 227 LAAIVPIAAAVP 12 6, 12 34.2 187.5 2.2 17 GGCSAPQTTCSN 12 6 51.6 8.3 −0.5 67 LDAEVPLADDVP 12 6, 12 34.2 130.0 0.3

TABLE 18 Proline Rigidity/ Sturctural rPeptide Position Flexibility Feature Hydropathy Group ID Sequences Length (PP) (II) (AI) (GARVY) Bending Peptides 692 PAPLPPVVILAV 12 1, 3, 5, 6 105.5 186.7 1.8 but Too High 69 PVAVLPPAALVP 12 1, 6, 7, 12 89.4 162.5 1.6 Flexibility 390 VPLLVPVVPVVP 12 2, 6, 9, 12 105.4 210.0 2.2 350 VPILVPVVPVVP 12 2, 6, 9, 12 121.5 210 0 2.2 331 VPVLVPLVPVVR 12 2, 6, 9, 12 105.4 210.0 2.2 9 VALVPAALILPP 12 5, 11, 12 89.4 203.3 2.1 68 VAPVLPPAPLVP 12 3, 6, 9, 12 105.5 162 5 1.6 349 VPVLVPVVPVVP 12 2, 6, 9, 12 121.5 201.6 2.2 937 VPVLVPLPVPVV 12 2, 6, 8, 10 121.5 210.0 2.2 938 VPVLLPVVVPVP 12 2, 6, 10, 12 121.5 210.0 2.2 329 LPVLVPVVPVVP 12 2, 6, 9, 12 121.5 210.0 2.2 49 VVPAAPAVPVVP 12 3, 6, 9, 12 121.5 145.8 1.7 772 LPVAPVIPIIVP 12 2, 5, 8, 12 79.9 210.8 2.1 210 ALIALPALPALP 12 6, 9, 12 89.4 195.8 1.8 28 AVPLLPLVPAVP 12 3, 6, 9, 12 89.4 186.8 1.8 693 AAPVLPVAVPIV 12 3, 6, 10 82.3 186.7 2.1 169 VALVAPALILAP 12 6, 12 73.4 211.7 2.4 29 VLPPLPVLPVLP 12 3, 4, 6, 9, 12 121.5 202.5 1.7 190 AAILAPAVIAPP 12 6, 11, 12 89.4 163.3 1.8

3-4-3. Peptides that do not Satisfy the Structural Features

New hydrophobic CPPs—aMTDs are consisted with only hydrophobic and aliphatic amino acids (A, V, L, I and P) with average ranges of the indexes—AI: 180 to 220 and GRAVY: 2.1 to 2.6 (Table 9). Based on the structural indexes, the peptides which contain an aromatic residue (W, F or Y) are shown in Table 19 and the peptides which are hydrophobic with non-aromatic sequences but have amino acids residue other than A, V, L, I, P or additional proline residues are designed (Table 20). Finally, hydrophilic and/or bending peptides which are consisted with non-aliphatic amino acids are shown in Table 21.

TABLE 19 Proline Rigidity/ Sturctural Length Position Flexibility Feature Hydropathy Group rPeptide ID Sequences (PP) (II) (AI) (GRAVY) Aromatic Peptides 30 WFFAGPIMLIWP 12 6, 12 9.2 105.1 1.4 (Aromatic Ring 33 AAAILAPAFLAV 12 7 57.3 171.7 2.4 Presences) 131 WIIAPVWLAWIA 12 5 51.6 179.2 1.9 922 WYVIFVLPLVVP 12 8, 12 41.3 194.2 2.2 71 FMWMWFPFMWYP 12 7, 12 71.3 0.0 0.6 921 IWWPVVLPLVVP 12 8, 12 41.3 194.2 2.2

TABLE 20 Proline Rigidity/ Sturctural rPeptide Position Flexibility Feature Hydropathy Group ID Sequences Length (PP) (II) (AI) (GARVY) Hydrophobic 436 VVMLVVPAVMLP 12 7, 12 57.3 194.2 2.6 but Non Aromatic 138 PPAALLAILAVA 12 1, 2 57.3 195.8 2.2 Peptides 77 PVALVLVALVAP 12 1, 12 41.3 219.2 2.5 577 MLMIALVPMIAV 12 8 18.9 195.0 2.7 97 ALLAAPPALLAL 12 6, 7 57.3 204.2 2.1 214 ALIVAPALMALP 12 6, 12 60.5 187.5 2.2 59 AVLAAPVVAALA 12 6 41.3 187.5 2.5 54 LAVAAPPVVALL 12 6, 7 57.3 203.3 2.3

TABLE 21 Proline Rigidity Structural Position Flexibility Feature Hydropathy Group rPeptide ID Sequences Length (PP) (II) (AI) (GRAVY) Hydrophilic Peptides 949 SGNSCOOCGNSS 12 None 41.7 0.0 −1.1 but Non Aliphatic 39 CYNTSPCTGCCY 12  6 52.5 0.0 0.0 19 YVSCCTYTNGSO 12 None 47.7 0.0 −1.0 947 CYYNOOSNNNNO 12 None 59.6 0.0 −2.4 139 TGSTNSPTCTST 12  7 53.4 0.0 −0.7 18 NYCCTPTTNGOS 12  6 47.9 0.0 −0.9 20 NYCNTCPTYGOS 12  7 47.4 0.0 −0.9 635 GSTGGSOONNOY 12 None 31.9 0.0 −1.9 40 TYNTSCTPGTCY 12  8 49.4 0.0 −0.6 57 ONNCNTSSOGGG 12 None 52.4 0.0 −1.6 159 CYSGSTSONOPP 12 11, 12 51.0 0.0 −1.3 700 GTSNTCOSNONS 12 None 19.1 0.0 −1.6 38 YYNOSTCGGOCY 12 None 53.3 0.0 −1.0

3-5. Summary of Newly Designed Peptides

Total of 457 sequences have been designed based on the critical factors. Designed potentially best aMTDs (hydrophobic, flexible, bending, aliphatic and 12-A/a length peptides) that do satisfy all range/feature of critical factors are 316. Designed rPeptides that do not satisfy at least one of the critical factors are 141 that no bending peptide sequences are 26; rigid peptide (11<40) sequences are 23; too much flexible peptides are 24; aromatic peptides (aromatic ring presences) are 27; hydrophobic, but non-aromatic peptides are 23; and hydrophilic, but non-aliphatic peptides are 18.

4. Preparation of Recombinant Report Proteins Fused to aMTDs and rPeptides

Recombinant proteins fused to aMTDs and others [rPeptides, reference hydrophobic CPP sequences (MTM and MTD)] were expressed in a bacterial system, purified with single-step affinity chromatography and prepared as soluble proteins in physiological condition. These recombinant proteins have been tested for the ability of their cell-permeability by utilizing flow cytometry and laser scanning confocal microscopy.

4-1. Selection of Cargo Protein for Recombinant Proteins Fused to Peptide Sequences

For clinical/non-clinical application, aMTD-fused cargo materials would be biologically active molecules that could be one of the following: enzymes, transcription factors, toxic, antigenic peptides, antibodies and antibody fragments. Furthermore, biologically active molecules could be one of these following macromolecules: enzymes, hormones, carriers, immunoglobulin, membrane-bound proteins, transmembrane proteins, internal proteins, external proteins, secreted proteins, virus proteins, native proteins, glycoproteins, fragmented proteins, disulfide bonded proteins, recombinant proteins, chemically modified proteins and prions. In addition, these biologically active molecules could be one of the following: nucleic acid, coding nucleic acid sequence, mRNAs, antisense RNA molecule, carbohydrate, lipids and glycolipids.

According to these pre-required conditions, a non-functional cargo to evaluate aMTD-mediated protein uptake has been selected and called as Cargo A (CRA) that should be soluble and non-functional. The domain (A/a 289 to 840; 184 A/a length) is derived from protein S (Genbank ID: CP000113.1).

4-2. Construction of Expression Vector and Preparation of Recombinant Proteins

Coding sequences for recombinant proteins fused to each aMTD are cloned NdeI (5′) and SalI (3′) in pET-28a(+) (Novagen, Darmstadt, Germany) from PCR-amplified DNA segments. PCR primers for the recombinant proteins fused to aMTD and rPeptides are represented by SEQ ID NOs: 481 to 797. Structure of the recombinant proteins is displayed in FIG. 1.

The recombinant proteins were forcedly expressed in E. coli BL21 (DE3) cells grown to an OD₆₀₀ of 0.6 and induced for 2 hours with 0.7 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The proteins were purified by Ni²+ affinity chromatography as directed by the supplier (Qiagen, Hilden, Germany) in natural condition. After the purification, purified proteins were dissolved in a physiological buffer such as DMEM medium.

 Potentially Best aMTDs (Hydrophobic, Flexible, Bending, 240   Aliphatic & Helical)

 Random Peptides  31   No Bending Peptides (No Proline at 5 or 6 and/or 12)  02   No Bending Peptides (No Central Proline)  01   Rigid Peptides (II < 50)  09   Too Much Flexible Peptides  09   Aromatic Peptides (Aromatic Ring Presences)  01   Hydrophobic, But Non-Aromatic Peptides  02   Hydrophilic, But Non-Aliphatic Peptides  07

4-3. Expression of aMTD- or Random Peptide (rP)-Fused Recombinant Proteins

One embodiment of the present invention also relates to the development method of aMTD sequences having cell-permeability. Using the standardized six critical factors, 316 aMTD sequences have been designed. In addition, 141 rPeptides are also developed that lack one of these critical factors: no bending peptides: i) absence of proline both in the middle and at the end of sequence or ii) absence of proline either in the middle or at the end of sequence, rigid peptides, too much flexible peptides, aromatic peptides (aromatic ring presence), hydrophobic but non-aromatic peptides, and hydrophilic but non-aliphatic peptides (Table 22).

These rPeptides are devised to be compared and contrasted with aMTDs in order to analyze structure/sequence activity relationship (SAR) of each critical factor with regard to the peptides' intracellular delivery potential. All peptide (aMTD or rPeptide)-containing recombinant proteins have been fused to the CRA to enhance the solubility of the recombinant proteins to be expressed, purified, prepared and analyzed.

These designed 316 aMTDs and 141 rPeptides fused to CRA were all cloned (FIG. 2) and tested for inducible expression in E. coli (FIG. 3). Out of these peptides, 240 aMTDs were inducibly expressed, purified and prepared in soluble form (FIG. 4). In addition, 31 rPeptides were also prepared as soluble form (FIG. 4).

To prepare the proteins fused to rPeptides, 60 proteins were expressed that were 10 out of 26 rPeptides in the category of no bending peptides (Table 16); 15 out of 23 in the category of rigid peptides [instability index (II)<40] (Table 17); 19 out of 24 in the category of too much flexible peptides (Table 18); 6 out of 27 in the category of aromatic peptides (Table 19); 8 out of 23 in the category of hydrophobic but non-aromatic peptides (Table 20); and 12 out of 18 in the category of hydrophilic but non-aliphatic peptides (Table 21).

4-4. Quantitative Cell-Permeability of aMTD-Fused Recombinant Proteins

The aMTDs and rPeptides were fluorescently labeled and compared based on the critical factors for cell-permeability by using flow cytometry and confocal laser scanning microscopy (FIGS. 5 to 8). The cellular uptake of the peptide-fused non-functional cargo recombinant proteins could quantitatively be evaluated in flow cytometry, while confocal laser scanning microscopy allows intracellular uptake to be assessed visually. The analysis included recombinant proteins fused to a negative control [rP38] that has opposite characteristics (hydrophilic and aromatic sequence: YYNQSTCGGQCY) to the aMTDs (hydrophobic and aliphatic sequences). Relative cell-permeability (relative fold) of aMTDs to the negative control was also analyzed (Table 23 and FIG. 9).

Table 23 shows the Comparison Analysis of Cell-Permeability of aMTDs with a Negative Control (A: rP38).

TABLE 23 Negative Control rP38 aMTD 19.6 ± 1.6* The Average of 240 aMTDs (Best: 164.2) *Relative Fold (aMTD in Geo Mean in its comparison to rP38)

Relative cell-permeability (relative fold) of aMTDs to the reference CPPs [B: MTM12 (AAVLLPVLLAAP), C: MTD85 (AVALLILAV)] was also analyzed (Tables 40 and 41)

Table 24 shows Comparison Analysis of Cell-Permeability of aMTDs with a Reference CPP (B: MTM12).

TABLE 24 MTM12 aMTD 13.1 ± 1.1* The Average of 240 aMTDs (Best: 109.9) *Relative Fold (aMTD in Geo Mean in its comparison to MTM12)

Table 25 shows the Comparison Analysis of Cell-Permeability of aMTDs with a Reference CPP (C: MTD85).

TABLE 25 MTD85 aMTD 6.6 ± 5* The Average of 240 aMTDs (Best: 55.5) *Relative Fold (aMTD in Geo Mean in its comparison to MTD85)

Geometric means of negative control (histidine-tagged rP38-fused CRA recombinant protein) subtracted by that of naked protein (histidine-tagged CRA protein) lacking any peptide (rP38 or aMTD) was standardized as relative fold of 1. Relative cell-permeability of 240 aMTDs to the negative control (A type) was significantly increased by up to 164 fold, with average increase of 19.6±1.6 (Tables 26 to 31).

TABLE 26 Proline Rigidity/ Sturctural Sequence Position Flexibility Feature Hydropathy Relative Ratio (Fold) ID Number aMTD Sequences Length (PP) (II) (AI) (GRAVY) A B C 1 899 AVVIALPAVVAP 12 7 57.3 195.0 2.4 164.2 109.9 55.5 2 908 VALALAPVVVAP 12 7 57.3 195.0 2.3 150.6 100.8 50.9 3 910 VAALLPAVVVAP 12 6 57.3 195.0 2.3 148.5 99.4 50.2 4 810 VIVLAAPALAAP 12 7 50.2 187.5 2.2 120.0 80.3 40.6 5 804 AVLAVVAPVVAP 12 8 57.3 186.7 2.4 105.7 70.2 35.8 6 321 IVAVALPALAVP 12 7 50.2 203.3 2.3 97.8 65.2 32.9 7 851 VLAVVLPAVALP 12 7 57.3 219.2 2.5 96.6 64.7 32.7 8 911 VALALPAVVVAP 12 6 57.3 195.2 2.3 84.8 56.2 28.7 9 852 VLAVAAPAVLLP 12 7 57.3 203.3 2.3 84.6 56.6 28.6 10 803 AIALAVPVLALP 12 7 57.3 211.7 2.4 74.7 50.0 25.3 11 888 ILAVVAIPAAAP 12 8 54.9 187.5 2.3 71.0 47.5 24.0 12 825 IVAVIVAPAVAP 12 8 43.2 195.0 2.6 69.7 46.6 23.6 13 895 AIIIVVPAIAAP 12 7 50.2 211.7 2.2 60.2 40.7 20.6 14 896 AILIVVAPIAAP 12 8 50.2 211.7 2.5 57.5 38.5 19.4 15 727 VALA1ALPAVLP 12 8 57.3 211.6 2.3 54.7 36.1 18.5 16 603 VLVALAAPVIAP 12 8 57.3 203.3 2.4 54.1 36.1 18.2 17 847 LVAIVVLPAVAP 12 8 50.2 219.2 2.6 50.2 33.4 12.9 18 826 LVALAAPIIAVP 12 7 41.2 211.7 2.4 49.2 32.2 16.6 19 724 VAVLAVLPALAP 12 8 57.3 203.2 2.3 47.5 31.8 16.1 20 563 ALAVIVVPALAP 12 8 50.2 203.3 2.4 47.1 31.4 15.9 21 811 AVVLAVPALAVP 12 7 57.3 195.0 2.3 46.5 31.1 15.7 22 831 IIVAVAPAAIVP 12 7 43.2 203.3 2.5 46.3 31.0 15.7 23 829 AALALVAPVIVP 12 8 50.2 203.3 2.4 44.8 30.0 15.2 24 891 ILAVAAIPAALP 12 8 54.9 195.2 2.2 44.7 29.9 15.1 25 905 AVIAVAPLVVAP 12 7 41.3 195.0 2.4 44.0 29.5 14.9 26 664 VAIALIVPALAP 12 8 50.2 211.7 2.4 43.6 29.1 14.7 27 124 IAVALPALIAAP 12 6 50.3 195.2 2.2 43.6 29.0 14.7 28 827 IAAVLAAPALVP 12 8 57.3 187.5 2.2 43.0 28.2 14.6 29 2 AAAVPLLAVVVP 12 5 41.2 195.0 2.4 40.9 27.2 13.8 30 385 IVAIAVPALVAP 12 7 50.2 203.3 2.4 38.8 25.9 13.1 31 828 IALLAAPIIAVP 12 7 41.3 220.0 2.4 36.8 24.6 12.4 32 806 LVALAVPAAVLP 12 7 57.3 203.3 2.3 36.7 24.2 12.4 33 845 AAVVIAPLLAVP 12 7 41.3 203.3 2.4 35.8 24.0 12.1 34 882 AIALVVPAVAVP 12 7 57.3 195.0 2.4 35.0 23.4 11.8 35 545 VVLVLAAPAAVP 12 8 57.2 195.0 2.3 34.6 23.1 11.7 36 161 AVIALPALIAAP 12 6 57.3 195.8 2.2 34.5 23.0 11.6 37 481 AIAIAIVPVALP 12 8 50.2 211.6 2.4 34.3 23.0 11.6 38 900 ALVAVIAPVVAP 12 8 57.3 195.0 2.4 34.3 22.9 11.6 39 223 AILAVPIAVVAP 12 6 57.3 203.2 2.4 33.0 22.1 11.2 40 824 LIIVAAAPAVAP 12 8 50.2 187.5 2.3 32.8 21.9 11.1 41 562 ALIAAIVPALVP 12 8 50.2 211.7 2.4 32.7 21.2 11.0 42 222 ALLIARAAVIAP 12 6 57.3 195.2 2.2 32.6 21.7 11.0 43 61 VAALPVLLAALP 12 5 57.3 211.7 2.3 31.2 20.2 10.5 44 582 VAVALIVPALAP 12 8 50.2 203.3 2.4 30.2 20.4 10.3 45 889 ILVAAAPIAALP 12 7 57.3 195.8 2.2 30.3 20.3 10.3 46 787 AVALVPVIVAAP 12 6 50.2 195.0 2.4 29.3 19.6 9.9 47 703 IVAVALVPALAP 12 8 50.2 203.3 2.4 29.2 19.5 9.9 48 705 IVAVALLPALAP 12 8 50.2 211.7 2.4 28.6 19.1 9.7 49 885 LVAIAPAVAVLP 12 6 57.3 203.3 2.4 28.3 19.0 9.6 50 3 AALLVPAAVLAP 12 6 57.3 187.5 2.1 27.0 18.0 9.1 51 601 AAILIAVPIAAP 12 8 57.3 195.8 2.3 26.8 17.9 9.0 52 843 AVLVLVAPAAAP 12 8 41.3 219.2 2.5 26.4 17.7 8.9 53 403 AAALVIPAAILP 12 7 54.9 195.8 2.2 25.2 16.8 8.5 54 544 IVALIVAPAAVP 12 8 43.1 203.3 2.4 23.4 15.6 7.9 55 522 ALLVIAVPAVAP 12 8 57.3 203.3 2.4 22.7 15.2 7.7

TABLE 27 Proline Rigidity/ Sturctural Sequence Position Flexibility Feature Hydropathy Relative Ration (Fold) ID Number aMTD Sequences Length (PP) (II) (AI) (GRAVY) A B C 56 805 LVLIAAAPIALP 12 8 41.3 220.0 2.4 22.3 14.9 7.6 57 464 AVVILVPLAAAP 12 7 57.3 203.3 2.4 22.3 14.9 7.5 58 405 LAAAVIPVAILP 12 7 54.9 211.7 2.4 22.2 14.8 7.5 59 747 VALLAIAPALAP 12 8 57.3 195.8 2.2 22.0 14.8 7.5 60 501 VIVALAVPALAP 12 8 50.2 203.3 2.4 21.5 14.4 7.3 61 661 AAILAPIVAALP 12 6 50.2 195.8 2.2 21.4 14.3 7.2 62 786 LVAIAPLAVLAP 12 6 41.3 211.7 2.4 21.2 14.2 7.2 63 625 ILAAAAAPLIVP 12 8 50.2 195.8 2.2 20.9 13.9 7.0 64 442 ALAALVPAVLVP 12 7 57.3 203.3 2.3 20.4 13.6 6.9 65 912 VALLAPAVVVAP 12 6 57.3 195.0 2.3 19.9 13.3 6.7 66 165 ALAVPVALAIVP 12 5 50.2 203.3 2.4 19.8 13.2 6.7 67 422 VVAILAPLLAAP 12 7 57.3 211.7 2.4 19.6 13.1 6.6 68 686 AALVAVLPVALP 12 8 57.3 203.3 2.3 19.5 13.1 6.6 69 343 IVAVALPALVAP 12 7 50.2 203.3 2.3 19.4 12.9 6.5 70 323 IVAVALPVALAP 12 7 50.2 203.3 2.3 19.1 12.8 6.4 71 461 IAAVIVPAVALP 12 7 50.2 203.3 2.4 19.0 12.7 6.4 72 21 AVALLPALLAVP 12 6 57.3 211.7 2.3 18.9 12.6 6.4 73 404 LAAAVIPAAILP 12 7 54.9 195.8 2.2 18.9 12.6 6.4 74 261 LVLVPLLAAAAP 12 5 41.3 211.6 2.3 18.5 12.3 6.2 75 524 AVALIVVPALAP 12 8 50.2 203.3 2.4 18.3 12.2 6.2 76 225 VAALLPAAAVLP 12 6 57.2 187.5 2.1 18.3 12.2 6.2 77 264 LAAAPVVIVIAP 12 5 50.2 203.3 2.4 18.2 12.1 6.1 78 1 AAALAPVVLALP 12 6 57.3 187.5 2.1 17.7 11.8 6.0 79 382 AAALVIPAILAP 12 7 54.9 195.8 2.2 17.7 11.8 6.0 80 463 AVAILVPLLAAP 12 7 57.3 211.7 2.4 17.6 11.7 5.9 81 322 VVAIVLPALAAP 12 7 50.2 203.3 2.3 17.6 11.7 5.9 82 503 AAIIIVLPAALP 12 8 50.2 220.0 2.4 17.6 11.8 5.9 83 870 VLVAAVLPIAAP 12 8 41.3 203.3 2.4 16.6 11.1 5.6 84 241 AAAVVPVLLVAP 12 6 57.3 195.0 2.4 16.6 11.0 5.6 85 726 LAVAIIAPAVAP 12 8 57.3 187.5 2.2 16.5 11.0 5.6 86 341 IVAVALPAVLAP 12 7 50.2 203.3 2.3 16.4 10.9 5.5 87 542 ALALIIVPAVAP 12 8 50.2 211.6 2.4 16.2 10.8 5.5 88 361 AVVIVAPAVIAP 12 7 50.2 195.0 2.4 16.0 10.7 5.4 89 224 ILAAVPIALAAP 12 6 57.3 195.8 2.2 15.8 10.6 5.3 90 482 ILAVAAIPVAVP 12 8 54.9 203.3 2.4 15.8 10.6 5.3 91 64 AIVALPVAVLAP 12 6 50.2 203.3 2.4 15.8 10.6 5.3 92 484 LAVVLAAPAIVP 12 8 50.2 203.3 2.4 15.6 10.4 5.3 93 868 VLVAAILPAAIP 12 8 54.9 211.7 2.4 14.9 10.0 5.0 94 541 LLALIIAPAAAP 12 8 57.3 204.1 2.1 14.8 9.9 5.0 95 666 AAIAIIAPAIVP 12 8 50.2 195.8 2.3 14.7 9.9 5.0 96 665 LAIVLAAPVAVP 12 8 50.2 203.3 2.3 14.7 9.9 5.0 97 363 AVLAVAPALIVP 12 7 50.2 203.3 2.3 14.7 9.8 4.9 98 242 AALLVPALVAAP 12 6 57.3 187.5 2.1 14.6 9.7 4.9 99 384 VIVAIAPALLAP 12 7 50.2 211.6 2.4 14.0 9.4 4.7 100 877 VAIIAVPAVVAP 12 7 57.3 195.0 2.4 14.0 9.4 4.7 101 863 AAVVLLPIIAAP 12 7 41.3 211.7 2.4 13.8 9.3 4.7 102 525 ALAIVVAPVAVP 12 8 50.2 195.0 2.4 13.8 9.2 4.7 103 875 AIAIVVPAVAVP 12 7 50.2 195.0 2.4 13.8 9.2 4.7 104 285 AIVLLPAAVVAP 12 6 50.2 203.3 2.4 13.3 8.9 4.5 105 281 ALIVLPAAVAVP 12 6 50.2 203.3 2.4 13.3 8.9 4.5 106 867 ALLVVIAPLAAP 12 8 41.3 211.7 2.4 13.2 8.8 4.4 107 766 IVVIAVAPAVAP 12 8 50.2 195.0 2.4 12.9 8.6 4.4 108 342 VIVALAPAVLAP 12 7 50.2 203.3 2.3 12.7 8.5 4.3 109 881 AALIVVPAVAVP 12 7 50.2 195.0 2.4 12.7 8.5 4.3 110 505 AIIIVIAPAAAP 12 8 50.2 195.8 2.3 12.4 8.3 4.2

TABLE 28 Se- Proline Rigidity/ Struc- Relative  quence Posi- Flexi- tural Hydro- Ratio ID tion bility Feature pathy (Fold) Number aMTD Sequences Length (PP) (II) (AI) (GRAVY) A B C 111 763 VAVLIAVPALAP 12 8 57.3 203.3 2.3 12.3 7.2 4.2 112 706 IVAVALLPAVAP 12 8 50.2 203.3 2.4 12.0 7.0 4.1 113 687 AILAVALPLLAP 12 8 57.3 220.0 2.3 12.0 7.0 4.1 114 643 LALVLAAPAIVP 12 8 50.2 211.6 2.4 11.8 7.9 4.0 115 282 VLAVAPALIVAP 12 6 50.2 203.3 2.4 11.8 7.9 4.0 116 543 LLAALIAPAALP 12 8 57.3 204.1 2.1 11.7 7.8 4.0 117 325 IVAVALPAVALP 12 7 50.2 203.3 2.3 11.7 7.8 4.0 118 846 IAVAVAAPLLVP 12 8 41.3 203.3 2.4 11.7 6.8 4.0 119 383 VIVALAPALLAP 12 7 50.2 211.6 2.3 11.6 7.7 3.9 120 381 VVAIVLPAVAAP 12 7 50.2 195.0 2.4 11.5 7.7 3.9 121 808 LVVLAAAPLAVP 12 8 41.3 203.3 2.3 11.5 7.6 3.9 122 865 AVLVIAVPAIAP 12 8 57.3 203.3 2.5 11.3 7.5 3.8 123 725 IAVLAVAPAVLP 12 8 57.3 203.3 2.3 11.2 7.5 3.8 124 844 VVALLAPLIAAP 12 7 41.3 211.8 2.4 11.2 7.5 3.8 125 897 AVIVPVAIIAAP 12 5 50.2 203.3 2.5 11.2 7.5 3.8 126 605 VIAAVLAPVAVP 12 8 57.3 195.0 2.4 11.0 7.4 3.7 127 744 AAVVIVAPVALP 12 8 50.2 195.0 2.4 11.0 7.3 3.7 128 221 AAILAPIVALAP 12 6 50.2 195.8 2.2 10.9 7.3 3.7 129 622 ALIVLAAPVAVP 12 8 50.2 203.3 2.4 10.6 7.1 3.6 130 401 AALAVIPAAILP 12 7 54.9 195.8 2.2 10.6 7.1 3.6 131 324 IVAVALPAALVP 12 7 50.2 203.3 2.3 10.3 6.9 3.5 132 878 IVALVAPAAVVP 12 7 50.2 195.0 2.4 10.3 6.9 3.5 133 302 LALAPALALLAP 12 5 57.3 204.2 2.1 10.2 6.8 3.4 134 685 ALLVAVLPAALP 12 8 57.3 211.7 2.3 10.2 5.9 3.4 135 848 AVAIVVLPAVAP 12 8 50.2 195.0 2.4 10.0 6.7 3.4 136 602 VIVALAAPVLAP 12 8 50.2 203.3 2.4 9.9 5.8 3.4 137 788 AIAVAIAPVALP 12 8 57.3 187.5 2.3 9.8 6.6 3.3 138 145 LLAVVPAVALAP 12 6 57.3 203.3 2.3 9.5 6.3 3.2 139  11 VVALAPALAALP 12 6 57.3 187.5 2.1 9.5 6.3 3.2 140 141 AVIVLPALAVAP 12 6 50.2 203.3 2.4 9.4 6.3 3.2 141 521 LAALIVVPAVAP 12 8 50.2 203.3 2.4 9.4 6.3 3.2 142 425 AVVAIAPVLALP 12 7 57.3 203.3 2.4 9.4 6.3 3.2 143 365 AVIVVAPALLAP 12 7 50.2 203.3 2.3 9.3 6.2 3.1 144 263 ALAVIPAAAILP 12 6 54.9 195.8 2.2 9.0 6.0 3.0 145 345 ALLIVAPVAVAP 12 7 50.2 203.3 2.3 8.9 5.9 3.0 146 850 LVIALAAPVALP 12 8 57.3 211.7 2.4 8.8 5.9 3.0 147 144 VLAIVPAVALAP 12 6 50.2 203.3 2.4 8.8 5.9 3.0 148 767 IVVAAVVPALAP 12 8 50.2 195.0 2.4 8.5 5.0 2.9 149 185 AALVLPLIIAAP 12 6 41.3 220.0 2.4 8.5 5.7 2.9 150 849 AVILLAPLIAAP 12 7 57.3 220.0 2.4 8.3 4.8 2.8 151 864 ALLVIAPAIAVP 12 7 57.3 211.7 2.4 8.2 4.8 2.8 152 162 AVVALPAALIVP 12 6 50.2 203.3 2.4 8.2 5.5 2.8 153 164 LAAVLPALLAAP 12 6 57.3 195.8 2.1 8.2 5.5 2.8 154 907 VAIALAPVVVAP 12 7 57.3 195.0 2.4 8.1 5.4 2.8 155 444 LAAALVPVALVP 12 7 57.3 203.3 2.3 8.1 5.4 2.7 156 443 ALAALVPVALVP 12 7 57.3 203.3 2.3 8.0 5.3 2.7 157 901 ALVAVLPAVAVP 12 7 57.3 195.0 2.4 7.7 5.1 2.6 158 887 VLAVAPAVAVLP 12 6 57.3 195.0 2.4 7.7 5.1 2.6 159 746 VAIIVVAPALAP 12 8 50.2 203.3 2.4 7.6 4.4 2.6 160 902 ALVAPLLAVAVP 12 5 41.3 203.3 2.3 7.6 5.1 2.6 161 565 VAIVLVAPAVAP 12 8 50.2 195.0 2.4 7.5 5.0 2.5 162 245 AAALAPVLALVP 12 6 57.3 187.5 2.1 7.5 5.0 2.5 163 743 AIAIALVPVALP 12 8 57.3 211.6 2.4 7.4 4.9 2.5 164 465 AVVILVPLAAAP 12 7 57.3 203.3 2.4 7.4 4.9 2.5 165 104 AVVAAPLVLALP 12 6 41.3 203.3 2.3 7.3 4.9 2.5

TABLE 29 Se- Proline Rigidity/ Struc- Relative  quence Posi- Flexi- tural Hydro- Ratio ID tion bility Feature pathy (Fold) Number aMTD Sequences Length (PP) (II) (AI) (GRAVY) A B C 166 707 IVALAVLPAVAP 12 8 50.2 203.3 2.4 7.3 4.9 2.5 167 872 VLAAAVLPLVVP 12 8 41.3 219.2 2.5 7.3 4.9 2.5 168 583 AVILALAPIVAP 12 8 50.2 211.6 2.4 7.3 4.8 2.4 169 879 AAIVLLPAVVVP 12 7 50.2 219.1 2.5 7.2 4.8 2.4 170 784 VAALPAVALVVP 12 5 57.3 195.0 2.4 7.1 4.7 2.4 171 893 VIAIPAILAAAP 12 5 54.9 195.8 2.3 7.0 4.7 2.4 172  13 AAALVPVVALLP 12 6 57.3 203.3 2.3 7.0 4.7 2.4 173 809 LIVLAAPALAAP 12 7 50.2 195.8 2.2 7.0 4.7 2.4 174 445 ALAALVPALVVP 12 7 57.3 203.3 2.3 6.9 4.6 2.3 175  81 AALLPALAALLP 12 5 57.3 204.2 2.1 6.9 4.6 2.3 176 667 LAVAIVAPALVP 12 8 50.2 203.3 2.3 6.9 4.6 2.3 177 906 AVIALAPVVVAP 12 7 57.3 195.0 2.4 6.8 4.6 2.3 178 483 ILAAAIIPAALP 12 8 54.9 204.1 2.2 6.8 4.5 2.3 179 485 AILAAIVPLAVP 12 8 50.2 211.6 2.4 6.8 4.5 2.3 180 421 AAILAAPLIAVP 12 7 57.3 195.8 2.2 6.7 4.5 2.3 181 585 ALIVAIAPALVP 12 8 50.2 211.6 2.4 6.6 4.4 2.2 182 424 AVVVAAPVLALP 12 7 57.3 195.0 2.4 6.6 4.4 2.2 183 364 LVAAVAPALIVP 12 7 50.2 203.3 2.3 6.5 4.3 2.2 184 402 ALAAVIPAAILP 12 7 54.9 195.8 2.2 6.4 4.3 2.2 185 462 IAAVLVPAVALP 12 7 57.3 203.3 2.4 6.3 4.2 2.1 186 265 VLAIAPLLAAVP 12 6 41.3 211.6 2.3 6.0 4.0 2.0 187 301 VIAAPVLAVLAP 12 6 57.3 203.3 2.4 6.0 4.0 2.0 188 183 LLAAPVVIALAP 12 6 57.3 211.6 2.4 6.0 4.0 2.0 189 243 AAVLLPVALAAP 12 6 57.3 187.5 2.1 5.9 3.9 2.0 190 664 ILIAIAIPAAAP 12 8 54.9 204.1 2.3 5.7 3.8 1.9 191 783 IVALVPAVAIAP 12 6 50.2 203.3 2.5 5.7 3.8 1.9 192 502 AIVALAVPVLAP 12 8 50.2 203.3 2.4 5.6 3.7 1.9 193 262 ALIAVPAIIVAP 12 6 50.2 211.6 2.4 5.5 3.7 1.9 194 683 LAIVLAAPAVLP 12 8 50.2 211.7 2.4 5.5 3.2 1.9 195 830 IALVAAPVALVP 12 7 57.3 203.3 2.4 5.3 3.5 1.8 196 764 AVALAVLPAVVP 12 8 57.3 195.0 2.3 5.0 3.4 1.7 197 807 AVALAVPALVLP 12 7 57.3 203.3 2.3 5.0 3.3 1.7 198 184 LAAIVPAIIAVP 12 6 50.2 211.6 2.4 4.8 3.2 1.6 199 305 IALAAPILLAAP 12 6 57.3 204.2 2.2 4.8 3.2 1.6 200 101 LVALAPVAAVLP 12 6 57.3 203.3 2.3 4.5 3.0 1.5 201 304 AIILAPIAAIAP 12 6 57.3 204.2 2.3 4.4 3.0 1.5 202 604 VALIAVAPAVVP 12 8 57.3 195.0 2.4 4.3 2.5 1.5 203 645 ALAVVALPAIVP 12 8 50.2 203.3 2.4 4.3 2.9 1.5 204 201 LALAVPALAALP 12 6 57.3 195.8 2.1 4.2 2.8 1.4 205 163 LALVLPAALAAP 12 6 57.3 195.8 2.1 4.1 2.4 1.4 206 832 AVAAIVPVIVAP 12 7 43.2 195.0 2.5 4.1 2.7 1.4 207 182 ALIAPVVALVAP 12 6 57.3 203.3 2.4 4.0 2.7 1.4 208  23 VVLVLPAAAAVP 12 6 57.3 195.0 2.4 4.0 2.6 1.3 209 105 LLALAPAALLAP 12 6 57.3 204.1 2.1 4.0 2.6 1.3 210 561 AAVAIVLPAVVP 12 8 50.2 195.0 2.4 3.9 2.6 1.3 211 765 AVALAVVPAVLP 12 8 57.3 195.0 2.3 3.8 2.2 1.3 212 684 AAIVLALPAVLP 12 8 50.2 211.7 2.4 3.5 2.1 1.2 213 143 AVLAVPAVLVAP 12 6 57.3 195.0 2.4 3.3 2.2 1.1 214 504 LIVALAVPALAP 12 8 50.2 211.7 2.4 3.3 2.2 1.1 215  22 AVVLVPVLAAAP 12 6 57.3 195.0 2.4 3.1 2.1 1.1 216   5 AAALLPVALVAP 12 6 57.3 187.5 2.1 3.1 2.1 1.0 217 283 AALLAPALIVAP 12 6 50.2 195.8 2.2 3.1 2.0 1.0 218  65 IAIVAPVVALAP 12 6 50.2 203.3 2.4 3.0 2.0 1.0 219 883 LAIVPAAIAALP 12 6 50.2 195.8 2.2 3.0 2.0 1.0 220 123 AAIIVPAALLAP 12 6 50.2 195.8 2.2 2.9 2.0 1.0

TABLE 30 Pro- Rigid-   Se- line ity/ Struc- Hydro- Relative quence Posi- Flexi- tural pathy Ratio ID tion bility Feature (GRA- (Fold) Number aMTD Sequences Length (PP) (II) (AI) VY) A B C 221 284 ALIAPAVALIVP 12 5 50.2 211.7 2.4  2.8  1.8 0.9 222 205 ALALVPAIAALP 12 6 57.3 195.8 2.2  2.6  1.7 0.9 223  42 VAALPVVAVVAP 12 5 57.3 186.7 2.4  2.5  1.7 0.8 224 121 AIVALPALALAP 12 6 50.2 195.8 2.2  2.5  1.7 0.8 225  25 IVAVAPALVALP 12 6 50.2 203.3 2.4  2.4  1.6 0.8 226  24 IALAAPALIVAP 12 6 50.2 195.8 2.2  2.3  1.6 0.8 227 204 LIAALPAVAALP 12 6 57.3 195.8 2.2  2.2  1.5 0.8 228  12 LLAAVPAVLLAP 12 6 57.3 211.7 2.3  2.2  1.5 0.7 229  43 LLAAPLVVAAVP 12 5 41.3 187.5 2.1  2.1  1.4 0.7 230 103 ALIAAPILALAP 12 6 57.3 204.2 2.2  2.1  1.4 0.7 231  82 AVVLAPVAAVLP 12 6 57.3 195.0 2.4  2.1  1.4 0.7 232   4 ALALLPVAALAP 12 6 57.3 195.8 2.1  2.0  1.3 0.7 233  85 LLVLPAAALAAP 12 5 57.3 195.8 2.1  1.9  1.3 0.7 234  63 AALLVPALVAVP 12 6 57.3 203.3 2.3  1.9  1.3 0.7 235  44 ALAVPVALLVAP 12 5 57.3 203.3 2.3  1.6  1.1 0.5 236  84 AAVAAPLLLALP 12 6 41.3 195.8 2.1  1.5  1.0 0.5 237  62 VALLAPVALAVP 12 6 57.3 203.3 2.3  1.4  0.9 0.5 238  83 LAVAAPLALALP 12 6 41.3 195.8 2.1  1.4  0.9 0.5 239 102 LALAPAALALLP 12 5 57.3 204.2 2.1  1.4  0.9 0.5 240 623 VAAAIALPAIVP 12 8 50.2 187.5 2.3  0.8  0.6 0.3 19.6 ± 13.1 ± 6.6 ±  1.6  1.1 0.5

Moreover, compared to reference CPPs (B type: MTM12 and C type: MTD85), novel 240 aMTDs averaged of 13±1.1 (maximum 109.9) and 6.6-0.5 (maximum 55.5) fold higher cell-permeability, respectively (Tables 26 to 31).

TABLE 31 Negative Control rP38 MTM12 MTD85 aMTD 19.6 ± 1.6* 13.1 ± 1.1* 6.6 ± 0.5* The Average of 240 aMTDs (Best: 164.2) (Best: 109.9) (Best: 55.5) *Relative Fold (aMTD in Geo Mean in its comparison to rP38, MTM12 or MTD85)

In addition, cell-permeabilities of 31 rPeptides have been compared with that of 240 aMTDs (0.3±0.04; Tables 32 and 33).

TABLE 32 Rela- Pro- Rigid- tive line ity/ Struc- Hydro- Ratio Posi- Flexi- tural pathy to Num- tion bility Feature (GRA- aMTD ber ID Sequence Length (PP) (II) (AI) VY) AVE  1 692 PAPLPPVVILAV 12  1,    105.5 186.7  1.8 0.74  3,  5,  6  2  26 AAIALAAPLAIV 12  8  18.1 204.2  2.5 0.65  3 113 PVAVALLIAVPP 12  1,   57.3 195.0  2.1 0.61 11,  12  4 466 IIAAAAPLAIIP 12  7,   22.8 204.2  2.3 0.52 12  5 167 VAIAIPAALAIP 12  6,   20.4 195.8  2.3 0.50 12  6  97 ALLAAPPALLAL 12  6,   57.3 204.2  2.1 0.41  7  7 390 VPLLVPVVPVVP 12  2,  105.4 210.0  2.2 0.41  6,   9,  12  8 426 AAALAIPLAIIP 12  7,    4.37 204.2  2.2 0.40 12  9 214 ALIVAPALMALP 12  6,   60.5 187.5  2.2 0.33 12 10  68 VAPVLPAAPLVP 12  3,  105.5 162.5  1.6 0.32  6,   9,  12 11  39 CYNTSPCTGCCY 12  6  52.5   0.0  0.0 0.29 12 934 LLLAPAAVVAAA 12  5  57.3 195.8  2.5 0.28 13 938 VPVLLPVVVPVP 12  2,  121.5 210.0  2.2 0.28  6,  10,  12 14 329 LPVLVPVVPVVP 12  2,  121.5 210.0  2.2 0.23  6,   9,  12 15 606 AAAIAAIPIIIP 12  8,    4.4 204.2  2.4 0.20 12 16  49 VVPAAPAVPVVP 12  3,  121.5 145.8  1.7 0.18  6,   9,  12 17 139 TGSTNSPTCTST 12  7  53.4   0.0 −0.7 0.17 18 772 LPVAPVIPIIVP 12  2,   79.9 210.8  2.1 0.16  5,   8,  12 19 921 IWWFVVLPLVVP 12  8,   41.3 194.2  2.2 0.14 12 20  66 AGVLGGPIMGVP 12  7,   35.5 121.7  1.3 0.13 12 21 693 AAPVLPVAVPIV 12  3,   82.3 186.7  2.1 0.13  6,  10 22  18 NYCCTPTTNGQS 12  6  47.9   0.0 −0.9 0.10 23  16 NNSCTTYTNGSQ 12 None  47.4   0.0 −1.4 0.08 24 227 LAAIVPIAAAVP 12  6,   34.2 187.5  2.2 0.08 12 25  17 GGCSAPQTTCSN 12  6  51.6   8.3 −0.5 0.08 26  67 LDAEVPLADDVP 12  6,   34.2 130.0  0.3 0.08 12 27 635 GSTGGSQQNNQY 12 None  31.9   0.0 −1.9 0.07 28  29 VLPPLPVLPVLP 12  3,  121.5 202.5  1.7 0.07  4,   6,   9,  12 29  57 QNNCNTSSQGGG 12 None  52.4   0.0 −1.6 0.06 30 700 GTSNTCQSNQNS 12 None  19.1   0.0 −1.6 0.05 31  38 YYNQSTCGGQCY 12 ND  53.8   0.0 −1.0 0.05 AVE 0.3 ± 0.04

TABLE 33 Relative Ratio to aMTD AVE* rPeptide 0.3 ± 0.04 The Average of 31 aMTDs *Out of 240 aMTDs, average relative fold of aMTD had been 19.6 fold compared to type A (rP38).

In summary, relatively cell-permeability of aMTDs has shown maximum of 164.0, 109.9 and 55.5 fold higher to rP38, MTM12 and MTD85, respectively. In average of total 240 aMTD sequences, 19.6±1.6, 13.1±1.1 and 6.6±0.5 fold higher cell-permeability are shown to the rP38, MTM12 and MTD85, respectively (Tables 26 to 31). Relative cell-permeability of negative control (rP38) to the 240 aMTDs is only 0.3±0.04 fold.

4-5. Intracellular Delivery and Localization of aMTD-Fused Recombinant Proteins

Recombinant proteins fused to the aMTDs were tested to determine their intracellular delivery and localization by laser scanning confocal microscopy with a negative control (rP38) and previous published CPPs (MTM12 and MTD85) as the positive control references. NIH3T3 cells were exposed to 10 uM of FITC-labeled protein for 1 hour at 37° C., and nuclei were counterstained with DAPI. Then, cells were examined by confocal laser scanning microscopy (FIG. 7). Recombinant proteins fused to aMTDs clearly display intracellular delivery and cytoplasmic localization (FIG. 7) that are typically higher than the reference CPPs (MTM12 and MTD85). The rP38-fused recombinant protein did not show internalized fluorescence signal (FIG. 7a ). In addition, as seen in FIG. 8, rPeptides (his-tagged CRA recombinant proteins fused to each rPeptide) display lower- or non-cell-permeability.

4-6. Summary of Quantitative and Visual Cell-Permeability of Newly Developed aMTDs

Histidine-tagged aMTD-fused cargo recombinant proteins have been greatly enhanced in their solubility and yield. Thus, FITC-conjugated recombinant proteins have also been tested to quantitate and visualize intracellular localization of the proteins and demonstrated higher cell-permeability compared to the reference CPPs.

In the previous studies using the hydrophobic signal-sequence-derived CPPs-MTS/MTM or MTDs, 17 published sequences have been identified and analyzed in various characteristics such as length, molecular weight, pI value, bending potential, rigidity, flexibility, structural feature, hydropathy, amino acid residue and composition, and secondary structure of the peptides. Based on these analytical data of the sequences, novel artificial and non-natural peptide sequences designated as advanced MTDs (aMTDs) have been invented and determined their functional activity in intracellular delivery potential with aMTD-fused recombinant proteins.

aMTD-fused recombinant proteins have promoted the ability of protein transduction into the cells compared to the recombinant proteins containing rPeptides and/or reference hydrophobic CPPs (MTM12 and MTD85). According to the results, it has been demonstrated that critical factors of cell-penetrating peptide sequences play a major role to determine peptide-mediated intracellular delivery by penetrating plasma membrane. In addition, cell-permeability can considerably be improved by following the rational that all satisfy the critical factors.

5. Structure/Sequence Activity Relationship (SAR) of aMTDs on Delivery Potential

After determining the cell-permeability of novel aMTDs, structure/sequence activity relationship (SAR) has been analyzed for each critical factor in selected some of and all of novel aMTDs (FIGS. 13a to 16 and Table 34).

TABLE 34 Rank of Rigidity/ Structural Relative Delivery Flexibility Feature Hydropathy Ratio (Fold) Amino Acid Composition Potential (II) (AI) (GRAVY) A B C A V I L  1~10 55.9 199.2 2.3 112.7 75.5 38.1 4.0 3.5 0.4 2.1 11~20 51.2 205.8 2.4 56.2 37.6 19.0 4.0 2.7 1.7 1.6 21~30 49.1 199.2 2.3 43.6 28.9 14.6 4.3 2.7 1.4 1.6 31~40 52.7 201.0 2.4 34.8 23.3 11.8 4.2 2.7 1.5 1.6 41~50 53.8 201.9 2.3 30.0 20.0 10.1 4.3 2.3 1.1 2.3 51~60 51.5 205.2 2.4 23.5 15.7 7.9 4.4 2.1 1.5 2.0 222~231 52.2 197.2 2.3 2.2 1.5 0.8 4.5 2.1 1.0 2.4 232~241 54.1 199.7 2.2 1.7 1.2 0.6 4.6 1.7 0.2 3.5

5-1. Proline Position:

In regards to the bending potential (proline position: PP), aMTDs with its proline at 7′ or 8′ amino acid in their sequences have much higher cell-permeability compared to the sequences in which their proline position is at 5′ or 6′ (FIGS. 14a and b and FIGS. 15a and b ).

5-2. Hydropathy:

In addition, when the aMTDs have GRAVY (Grand Average of Hydropathy) ranging in 2.1 to 2.2, these sequences display relatively lower cell-permeability, while the aMTDs with 2.3 to 2.6 GRAVY are shown significantly higher one (FIGS. 14c and d and FIGS. 15c and d ).

5-3. rPeptide SAR:

To the SAR of aMTDs, rPeptides have shown similar SAR correlations in the cell-permeability, pertaining to their proline position (PP) and hydropathy (GRAVY). These results confirm that rPeptides with high GRAVY (2.4 to 2.6) have better cell-permeability (FIG. 16).

5-4. Analysis of Amino Acid Composition:

In addition to proline position and hydropathy, the difference of amino acid composition is also analyzed. Since aMTDs are designed based on critical factors, each aMTD-fused recombinant protein has equally two proline sequences in the composition. Other hydrophobic and aliphatic amino acids—alanine, isoleucine, leucine and valine—are combined to form the rest of aMTD peptide sequences.

Alanine:

In the composition of amino acids, the result does not show a significant difference by the number of alanine in terms of the aMTD's delivery potential because all of the aMTDs have three to five alanines. However, in the sequences, four alanine compositions show the most effective delivery potential (geometric mean) (FIGS. 13a and b ).

Leucine and Isoleucine:

Also, the compositions of isoleucine and leucine in the aMTD sequences show inverse relationship between the number of amino acid (I and L) and delivery potential of aMTDs. Lower number of isoleucine and leucine in the sequences tends to have higher delivery potential (geometric mean) (FIGS. 13a to 13d ).

Valine:

Conversely, the composition of valine of aMTD sequences shows positive correlation with their cell-permeability. When the number of valine in the sequence is low, the delivery potential of aMTD is also relatively low (FIGS. 13c and d ).

Ten aMTDs having the highest cell-permeability are selected (average geometric mean: 2584±126). Their average number of valine in the sequences is 3.5; 10 aMTDs having relatively low cell-permeability (average geometric mean: 80±4) had average of 1.9 valine amino acids. The average number of valine in the sequences is lowered as their cell-permeability is also lowered as shown in FIGS. 13c and 13d . Compared to higher cell-permeable aMTDs group, lower sequences had average of 1.9 in their valine composition. Therefore, to obtain high cell-permeable sequence, an average of 2-4 valines should be composed in the sequence.

5-5. Conclusion of SAR Analysis:

As seen in FIG. 15, all 240 aMTDs have been examined for these associations of the cell-permeability and the critical factors: bending potential (PP), rigidity/flexibility (II), structure feature (AI), and hydropathy (GRAVY), amino acid length and composition. Through this analysis, cell-permeability of aMTDs tends to be lower when their central proline position is at 5′ or 6′ and GRAVY is 2.1 or lower (FIG. 15). Moreover, after investigating 10 higher and 10 lower cell-permeable aMTDs, these trends are clearly shown to confirm the association of cell-permeability with the central proline position and hydropathy.

6. Experimental Confirmation of Index Range/Feature of Critical Factors

The range and feature of five out of six critical factors have been empirically and experimentally determined that are also included in the index range and feature of the critical factors initially proposed before conducting the experiments and SAR analysis. In terms of index range and feature of critical factors of newly developed 240 aMTDs, the bending potential (proline position: PP), rigidity/flexibility (Instability Index: II), structural feature (Aliphatic Index: AI), hydropathy (GRAVY), amino acid length and composition are all within the characteristics of the critical factors derived from analysis of reference hydrophobic CPPs.

Therefore, our hypothesis to design and develop new hydrophobic CPP sequences as advanced MTDs is empirically and experimentally proved and demonstrated that critical factor-based new aMTD rational design is correct.

TABLE 35 Summarized Critical Factors of aMTD Newly Designed Analysis of CPPs Experimental Results Critical Factor Range Range Bending Potential Proline presences in the Proline presences in the (Proline middle (5′, 6′, 7‘ or 8′) middle (5′, 6′, 7‘ or 8′) and Position: PP) and at the end of peptides at the end of peptides Rigidity/Flexibility 40-60 41.3-57.3 (Instability Index: II) Structural Feature 180-220 187.5-220.0 (Aliphatic Index: Al) Hydropathy 2.1-2.6 2.2-2.6 (Grand Average of Hydropathy GRAVY) Length  9-13 12 (Number of Amino Acid) Amino acid A, V, I, L, P A, V, I, L, P Composition 7. Discovery and Development of Protein-Based New Biotherapeutics with MITT Enabled by aMTDs for Protein Therapy

240 aMTD sequences have been designed and developed based on the critical factors. Quantitative and visual cell-permeability of 240 aMTDs (hydrophobic, flexible, bending, aliphatic and 12 ala-length peptides) are all practically determined.

To measure the cell-permeability of aMTDs, rPeptides have also been designed and tested. As seen in FIGS. 13a through 15d , there are vivid association of cell-permeability and the critical factors of the peptides. Out of these critical factors, we are able to configure that the most effective cell-permeable aMTDs have the amino acid length of 12; composition of A, V, L, I and P; multiple proline located at either 7′ or 8′ and at the end (12′); instability index ranged of 41.3 to 57.3; aliphatic index ranged of 187.5 to 220.0; and hydropathy (GRAVY) ranged of 2.2 to 2.6.

These examined critical factors are within the range that we have set for our critical factors; therefore, we are able to confirm that the aMTDs that satisfy these critical factors have relatively high cell-permeability and much higher intracellular delivery potential compared to reference hydrophobic CPPs reported during the past two decades.

It has been widely evident that many human diseases are caused by proteins with deficiency or over-expression that causes mutations such as gain-of-function or loss-of-function. If biologically active proteins could be delivered for replacing abnormal proteins within a short time frame, possibly within an hour or two, in a quantitative manner, the dosage may be regulated depending on when and how proteins may be needed. By significantly improving the solubility and yield of novel aMTD according to one embodiment of the present invention (Table 31), one could expect its practical potential as an agent to effectively deliver therapeutic macromolecules such as proteins, peptides, nucleic acids, and other chemical compounds into live cells as well as live mammals including human. Therefore, newly developed MITT utilizing the pool (240) of novel aMTDs can be used as a platform technology for discovery and development of protein-based biotherapeutics to apprehend intracellular protein therapy after determining the optimal cargo-aMTD relationship.

8. Novel Hydrophobic CPPs—aMTDs for Development of iCP-RF Recombinant Proteins

8-1. Selection of aMTD for Cell-Permeability

From 240 aMTDs, 8 aMTDs were selected and used for the construction of iCP-RF recombinant proteins. 8 aMTDs used are shown in the following Table 36.

Various hydrophobic CPP have been used to enhance the delivery of protein cargoes to mammalian cells and tissues.

TABLE 36 SEQ ID aMTD Amino Acid NO ID Sequences  39 161 AVIALPALIAAP  43 165 ALAVPVALAIVP  84 363 AVLAVAPALIVP  96 405 LAAAVIPVAILP 131 563 ALAVIVVPALAP 223 889 ILVAAAPIAALP 226 895 AIIIVVPAIAAP 233 904 AVLAVVAPVVAP

8-2. Selection of Solubilization Domain (SD) for Structural Stability

Recombinant cargo (OCT4, SOX2, CMYC, KLF4, NANOG, LIN28 and ZSCAN4) proteins fused to hydrophobic CPP could be expressed in bacteria system, purified with single-step affinity chromatography, but protein dissolved in physiological buffers (e.q. PBS, DMEM or RPMI1640 etc.) was highly insoluble and had extremely low yield as a soluble form. Therefore, an additional non-functional protein domain (solubilization domain: SD) has been applied to fuse with the recombinant protein for improving the solubility, yield and eventually cell and tissue permeability.

According to the specific aim, the selected domains are SDA to SDF (Table 37). The aMTD/SD-fused RF recombinant proteins have been determined for their stability.

The solubilization domains (SDs) and aMTDs have greatly influenced in increasing solubility/yield and cell-/tissue-permeability of the protein. Therefore, we have developed highly soluble and highly stable RF recombinant protein fused with SD (SDA and/or SDB) and aMTDs.

Table 37 shows the Characteristics of Solubilization Domains.

TABLE 37 Protein Instability SD Genbank ID Origin (kDa) pI Index (II) GRAVY A CP000113.1 Bacteria 23 4.6 48.1 −0.1 B BC086945.1 Rat 11 4.9 43.2 −0.9 C CP012127.1 Human 12 5.8 30.7 −0.1 D CP012127.1 Bacteria 23 5.9 26.3 −0.1 E CP011550.1 Human 11 5.3 44.4 −0.9 F NG_034970 Human 34 7.1 56.1 −0.2

8-3. Construction of Expression Vector

5 different types of recombinant proteins with or without the aMTD and solubilization domains (SDs) for reprogramming factor (RF) protein were designed. Protein structures were labeled as follows: (1) a RF protein fused with His-tag, (2) a RF protein fused with His-tag, NLS and aMTD, (3) a RF protein fused with His-tag, NLS, aMTD and solubilization domain B (SDB), (4) a RF protein fused with His-tag, NLS, aMTD, solubilization domain A (SDA) and two solubilization domain B (SDB), and (5) a RF protein fused with His-tag, NLS, three solubilization domain A (SDA) and two solubilization domain B (SDB), (FIGS. 18, 20, 22, 24, 26, 28 and 30). Among them, (3) to (5) structures were used as candidate proteins having the biological efficacy of iCP-RF recombinant protein, and (1) and (2) were used as control groups (Non-CP RF) with respect to (3) to (5).

8-4. Preparation of RF Recombinant Proteins

The RF recombinant proteins (OCT4, SOX2, CMYC, KLF4, NANOG, LIN28 and ZSCAN4) were successfully induced by adding IPTG and purified (FIGS. 19, 21, 23, 25, 27, 29 and 31, top). The solubility and yield of the RF recombinant proteins were determined.

Solubility will be scored on a 5-point scale ranging from highly soluble proteins with little tendency to precipitate (*****) to largely insoluble proteins (*) by measuring their turbidity (A450). Yield (mg/L) in physiological buffer condition of each recombinant protein will also be determined.

We observed a significant increase of solubility of RF protein fused with SDB on C-terminus (HNM₅₆₃OSB, HNM₅₆₃MSB, HNM₁₆₁NSB, HNM₅₆₃LSB and HNM₅₆₃ZSB) and RF protein fused with both SDAs and SDBs on C-/N-terminus (HNM₅₆₃SASSASBSASB and HNM₅₆₃SAKSASBSASB), which were compared to a RF protein only or RF protein fused with aMTD on N-terminus (FIGS. 19, 21, 23, 25, 27, 29 and 31, bottom). And, we observed that yield and solubility of RF protein fused with SDB or both SDA and SDB on N-/C-terminus were greatly improved. The results suggested that the RF recombinant proteins fused with SD displayed a significant improvement of solubility and yields.

Further, solubility and yield of the RF recombinant proteins fused with different aMTDs (FIGS. 32, 34, 36 and 38, bottom) were measured. We observed that increase of both yield and solubility of SOX2 protein fused with aMTD₅₆₃, which were compared to a SOX2 protein fused with aMTD₁₆₁, aMTD₁₆₅, aMTD₃₆₃, aMTD₄₀₅, aMTD₈₈₉ and aMTD₉₀₄ (FIG. 33, bottom), and increase of both yield and solubility of NANOG protein fused with aMTD₁₆₁, which were compared to a NANOG protein fused with aMTD₄₀₅, aMTD₈₈₉, aMTD₈₉₅ and aMTD₉₀₄ (FIG. 35, bottom).

As a result, iCP-RF (OCT4, SOX2, CMYC, KLF4, NANOG, LIN28 and ZSCAN4) recombinant proteins were selected by comparing the solubility and yield between the RF recombinant proteins.

9. Determination of Cell-Permeability of iCP-RF Recombinant Proteins

In the cell-permeability of iCP-RF recombinant proteins (OCT4, SOX2, CMYC, KLF4 and LIN28) was investigated.

RF recombinant proteins were labeled fluorescence dye, FITC (fluorescein isothiocyanate), then cell permeability of the RF recombinant proteins was evaluated in RAW 264.7 cells and NIH3T3 cells. The RAW 264.7 cells and NIH3T3 cells were cultured in DMEM media containing 10% fetal bovine serum (FBS) and 500 mg/ml of 5% penicillin/streptomycin (P/S). After the culture, the cells were treated with Trypsin/EDTA for removal of the remained FITC on the cell membranes of the RAW 264.7 cells and the NIH3T3 cells, and washed with cold PBS three times.

The RAW 264.7 cells analyzed by FACS (fluorescence-activated cell sorting) showed a gain in fluorescence, indicative of the presence of FITC-labeled proteins as compared with control that only FITC or diluent. For FACS analysis, the cells (1×10⁴) were analyzed using the CellQues Pro cytometric analysis software (FACS Calibur, Beckton-Dickinson, San Diego Calif., USA). Cell permeability of each iCP-RF recombinant protein fused with aMTD/SD was examined (FIG. 39).

In the NIH3T3 cells, DNAs were stained with DAPI (4′,6-diamidino-2-phenylindole) to distinguish intracellular localization of the RF recombinant proteins, and intranuclear delivery and cell-permeability of the RF recombinant proteins were examined by confocal laser microscopy (FIG. 40). The original shape of the cells and both FITC and DAPI staining of the cells were observed by means of a confocal laser microscope using a Nomarski filter.

As a result, aMTD/SD-fused iCP-RF recombinant proteins have cell-permeability and are delivered to the nucleus.

10. Determination of Biological Activity of iCP-RF Recombinant Proteins

Reprogramming factors (RFs) (OCT4, SOX2, CMYC, KLF4, NANOG and LIN28) are transcription factors which bind to target genes to activate or inhibit transcription of the genes. Biological activity of the iCP-RF recombinant proteins was determined by measuring activities of the target genes which occur upon binding of the RF proteins and the genes. A luciferase vector that expresses luciferase when the iCP-RF recombinant protein binds to the target gene was constructed (FIG. 41a ). The luciferase vector was constructed, based on a pGL3 basic vector (Genscript, USA). Promoters containing 4 repeats of the binding sites of OCT4, SOX2, KLF4, CMYC, NANOG and LIN28 were synthesized. The vector and the promoter were digested using KpnI/HindIII restriction enzymes, and followed by ligation using T4 ligase.

As a result, the iCP-RF recombinant proteins delivered into the cells or nucleus exhibit a biological activity by binding to the DNA binding site of the luciferase promoter to express luciferase.

11. Determination of Formation of iPSC-Like Colony by iCP-RF Recombinant Proteins

Generation of iPSCs by treatment from somatic cells with the iCP-RF recombinant proteins was confirmed. An effective preparation method of iPSCs was determined by controlling combination, concentration, treatment duration, and treatment time of the iCP-RF recombinant proteins (OCT4, SOX2, KLF4, CMYC, LIN28, NANOG and ZSCAN4) (FIGS. 47 to 51, top). Since iPSCs express alkaline phosphatase (AP) on their surface, AP staining was performed. Further, stem cell-specific biomarkers, OCT4 and TRA-1-81 in iPSCs were examined by immunofluorescence staining analysis (FIG. 52, top).

As a result, the iCP-RF recombinant proteins have reprogramming activity for a somatic cell, and therefore, they are able to induce dedifferentiation of terminally differentiated somatic cells to iPSCs.

12. Summary

According to one embodiment of the present invention, cell-permeable RF recombinant proteins have been designed and developed with the aMTD and SDs. All RF recombinant proteins fused with aMTD/SD and control recombinant proteins lacking both aMTD and SD have been confirmed for their quantitative, visual cell-permeability and biological activity in vitro. Consequently, the RF recombinant proteins fused with aMTD/SD has relatively high solubility and yield, and the optimized structure of the RF recombinant proteins was determined. The optimal aMTD was also determined for the high yield and solubility of the RF recombinant proteins. The RF proteins fused with optimal aMTD and SD were improved cell-permeable RF (iCP-RF) recombinant proteins. It was confirmed that these iCP-RF recombinant proteins induce reprogramming of terminally differentiated somatic cells into iPSCs in a combination of iCP-OCT4, iCP-SOX2, iCP-KLF4, iCP-CMYC, iCP-LIN28, iCP-NANOG and iCP-ZSCAN4.

The following examples are presented to aid practitioners of the invention, to provide experimental support for the invention, and to provide model protocols. In no way are these examples to be understood to limit the embodiment.

Example 1. Development of Novel Advanced Macromolecule Transduction Domain (aMTD)

H-regions of signal sequences (HOURSP)-derived CPPs (MTS/MTM and MTD) do not have a common sequence, a sequence motif, and/or a common structural homologous feature. According to one embodiment of the present invention, the aim is to develop improved hydrophobic CPPs formatted in the common sequence and structural motif that satisfy newly determined ‘critical factors’ to have a ‘common function,’ to facilitate protein translocation across the plasma membrane with similar mechanism to the analyzed CPPs.

The structural motif as follows: X1-X2-X3-X4-U5-U6-U7-U8-X9-X10-X11-P

X: A, V, L or I

P: Proline

U: Proline at any one or X

In Table 9, universal common sequence/structural motif is provided as follows. The amino acid length of the peptides according to one embodiment of the present invention ranges from 9 to 13 amino acids, mostly 12 amino acids, and their bending potentials are dependent with the presence and location of proline in the middle of sequence (i.e., U5, U6, U7, or U8) and at the end of peptide (at 12′) for recombinant protein bending. Instability index (II) for rigidity/flexibility of aMTDs is 11<40, grand average of hydropathy (GRAVY) for hydropathy is around 2.2, and aliphatic index (AI) for structural features is around 200 (Table 9). Based on these standardized critical factors, new hydrophobic peptide sequences, namely advanced macromolecule transduction domain peptides (aMTDs), according to one embodiment of the present invention have been developed and summarized in Tables 10 to 15.

Example 2. Construction of Expression Vectors for Recombinant Proteins Fused to aMTDs

Our newly developed technology has enabled us to expand the method for making cell-permeable recombinant proteins. The expression vectors were designed for histidine-tagged CRA proteins fused with aMTDs or rPeptides. To construct expression vectors for recombinant proteins, polymerase chain reaction (PCR) had been devised to amplify each designed aMTD or rPeptide fused to CRA.

The PCR reactions (100 ng genomic DNA, 10 pmol each primer, each 0.2 mM dNTP mixture, 1× reaction buffer and 2.5 U Pfu(+) DNA polymerase (Doctor protein, Korea) was digested on the restriction enzyme site between Nde 1(5′) and Sal I (3′) involving 35 cycles of denaturation (95° C.), annealing (62° C.), and extension (72° C.) for 30 seconds each. For the last extension cycle, the PCR reactions remained for 5 minutes at 72° C. Then, they were cloned into the site of pET-28a(+) vectors (Novagen, Madison, Wis., USA). DNA ligation was performed using T4 DNA ligase at 4° C. overnight. These plasmids were mixed with competent cells of E. coli DH5-alpha strain on the ice for 10 minutes. This mixture was placed on the ice for 2 minutes after it was heat shocked in the water bath at 42° C. for 90 seconds. Then, the mixture added with LB broth media was recovered in 37° C. shaking incubator for 1 hour. Transformant was plated on LB broth agar plate with kanamycin (50 ug/mL) (Biopure, Johnson City, Tenn., USA) before incubating at 37° C. overnight. From a single colony, plasmid DNA was extracted, and after the digestion of Nde I and Sal I restriction enzymes, digested DNA was confirmed at 645 bp by using 1.2% agarose gels electrophoresis (FIG. 2). PCR primers for the CRA recombinant proteins fused to aMTD and random peptides (rPeptide) are summarized in Tables 23 to 30. Amino acid sequences of aMTD and rPeptide primers are shown in Tables 31 to 38.

Example 3. Inducible Expression, Purification and Preparation of Recombinant Proteins Fused to aMTDs and rPeptides

To express recombinant proteins, pET-28a(+) vectors for the expression of CRA proteins fused to a negative control [rPeptide 38 (rP38)], reference hydrophobic CPPs (MTM₁₂ and MTD₈₅) and aMTDs were transformed in E. coli BL21 (DE3) strains. Cells were grown at 37° C. in LB medium containing kanamycin (50 ug/ml) with a vigorous shaking and induced at OD₆₀₀=0.6 by adding 0.7 mM IPTG (Biopure) for 2 hours at 37° C. Induced recombinant proteins were loaded on 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue (InstantBlue, Expedeon, Novexin, UK) (FIG. 3).

The E. coli cultures were harvested by centrifugation at 5,000×rpm for 10 minutes, and the supernatant was discarded. The pellet was re-suspended in the lysis buffer (50 mM NaH₂PO₄, 10 mM Imidazol, 300 mM NaCl, pH 8.0). The cell lysates were sonicated on ice using a sonicator (Sonics and Materials, Inc., Newtown, Conn., USA) equipped with a probe.

After centrifuging the cell lysates at 5,000×rpm for 10 minutes to pellet the cellular debris, the supernatant was incubated with lysis buffer-equilibrated Ni-NTA resin (Qiagen, Hilden, Germany) gently by open-column system (Bio-rad, Hercules, Calif., USA). After washing protein-bound resin with 200 ml wash buffer (50 mM NaH₂PO₄, 20 mM Imidazol, 300 mM NaCl, pH 8.0), the bounded proteins were eluted with elution buffer (50 mM NaH₂PO₄, 250 mM Imidazol, 300 mM NaCl, pH 8.0).

Recombinant proteins purified under natural condition were analyzed on 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue (FIG. 4). All of the recombinant proteins were dialyzed for 8 hours and overnight against physiological buffer, a 1:1 mixture of cell culture medium (Dulbecco's Modified Eagle's Medium: DMEM, Hyclone, Logan, Utah, USA) and Dulbecco's phosphate buffered saline (DPBS, Gibco, Grand Island, N.Y., USA). From 316 aMTDs and 141 rPeptides cloned, 240 aMTD- and 31 rPeptide-fused recombinant proteins were induced, purified, prepared and analyzed for their cell-permeability.

Example 4. Determination of Quantitative Cell-Permeability of Recombinant Proteins

For quantitative cell-permeability, the aMTD- or rPeptide-fused recombinant proteins were conjugated to fluorescein isothiocyanate (FITC) according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, Mo., USA). RAW 264.7 cells were treated with 10 uM FITC-labeled recombinant proteins for 1 hour at 37° C.° C., washed three times with cold PBS, treated with 0.25% tripsin/EDTA (Sigma-Aldrich, St. Louis, Mo.) for 20 minutes at 37° C.° C. to remove cell-surface bound proteins. Cell-permeability of these recombinant proteins were analyzed by flow cytometry (Guava, Millipore, Darmstadt, Germany) using the FlowJo cytometric analysis software (FIGS. 5 to 6). The relative cell-permeability of aMTDs were measured and compared with the negative control (rP38) and reference hydrophobic CPPs (MTM12 and MTD85) (Table 31).

Example 5. Determination of Cell-Permeability and Intracellular Localization of Recombinant Proteins

For a visual reference of cell-permeability, NIH3T3 cells were cultured for 24 hours on coverslip in 24-wells chamber slides, treated with 10 uM FITC-conjugated recombinant proteins for 1 hour at 37° C., and washed three times with cold PBS. Treated cells were fixed in 4% paraformaldehyde (PFA, Junsei, Tokyo, JP) for 10 minutes at room temperature, washed three times with PBS, and mounted with VECTASHIELD Mounting Medium (Vector laboratories, Burlingame, Calif., USA), and counter stained with DAPI (4′,6-diamidino-2-phenylindole). The intracellular localization of the fluorescent signal was determined by confocal laser scanning microscopy (LSM700, Zeiss, Germany; FIGS. 7 and 8).

Example 6. Expression RF Recombinant Proteins

<6-1> Construction of Expression Vectors for Recombinant Proteins

Our newly developed technology, aMTD-based MITT, has enabled us to improve the method for developing cell-permeable recombinant proteins. The expression vectors were designed for RF proteins (OCT4, SOX2, CMYC, KLF4, NANOG, LIN28 and ZSCAN4) fused with aMTD/SDs (HNM#SB, HNMSA#SBSB and HNMSA#SASBSASB) and control proteins without aMTD and/or SD (H# and HNM#). To acquire expression vectors for RF recombinant proteins, polymerase chain reaction (PCR) had been devised to amplify these recombinant proteins.

The PCR reactions (100 ng genomic DNA, 10 pmol each primer, each 0.2 mM dNTP mixture, 1× reaction buffer and 2.5 U Pfu(+) DNA polymerase (Doctor Protein, Korea)) was digested on the different restriction enzyme site involving 40 cycles of denaturation (95° C.), annealing (58° C.), and extension (72° C.) for 30 seconds each. For the last extension cycle, the PCR reactions remained for 10 minutes at 72° C.

Histidine-tagged human RF proteins were separately constructed by amplifying the original gene cDNA for each proteins, including OCT4 (360 aa), SOX2 (317 aa), CMYC (439 aa), KLF4 (470 aa), NANOG (305 aa), LIN28 (209 aa) and ZSCAN4 (433 aa), using their specific primers (Tables 28 to 44), for aMTD/SD fused to RF proteins. The PCR products are cleaved with NdeI and SalI, then ligated into 6×His expression vector, pET-28a(+) (Novagen, USA). The amino acid sequences and cDNA of human RFs, independently, were shown in SEQ ID NOs: 816 to 822 and SEQ ID NOs: 823 to 829. For OCT4 or CMYC recombinant protein, NLS/aMTD-OCT4 or NLS/aMTD-CMYC was ligated into the NdeI and BamHI sites in pET-28(a) vector where SDB was located between the BamHI and SalI sites. For SOX2 or KLF4 recombinant protein, NLS/aMTD-SDA was ligated into the NdeI and BamHI sites in pET-28(a) vector where SOX2 or KLF4 was located between the BamHI and HindIII sites. SA/SB/SA/SB was located between the HindIII and XhoI sites. For NANOG recombinant protein, NLS/aMTD-NANOG was ligated into the NdeI and Sail sites in pET-28(a) vector where SDB was located between the Sail and XhoI sites. For LIN28 recombinant protein, NLS/aMTD-LIN28 was ligated into the EcoRI and SalI sites in pET-28(a) vector where SDB was located between the Sail and XhoI sites. For ZSCAN4 recombinant protein, NLS/aMTD-ZSCAN4 was ligated into the EcoRI and Sal sites in pET-28(a) vector where SDB was located between the SalI and NotI sites. DNA ligations, independently, were performed using T4 DNA ligase (NEB, USA) at 4° C. overnight.

These plasmids were mixed with competent cells of E. coli(BL21(DE3) codon plus RIL) strain (Agilent, USA) on the ice for 10 minutes. This mixture was placed on the ice for 2 minutes after it was heat shocked in the water bath at 42° C. for 90 seconds. Then, the mixture added with LB broth media (ELPIS, Korea) was recovered in 37° C. shaking incubator for 1 hour. Transformant was plated on LB broth agar plate with kanamycin (50 ug/mL) with a vigorous shaking and induced with 0.7 mM IPTG (Biopure, Johnson, Tenn.) at OD₆₀₀=0.6 before incubating at 37° C. overnight. From a single colony, plasmid DNA was extracted, and after the digestion of BamHI and HindIII restriction enzymes (NEB, USA), digested DNA was confirmed by using 1.2% agarose gels electrophoresis (FIGS. 17a to 17g ).

As shown in FIGS. 17a to 17g , it was confirmed that the RF recombinant proteins (OCT4, SOX2, KLF4, CMYC, NANOG, LIN28 and ZSCAN4) were expressed from the respective recombinant expression vectors.

TABLE 38 Cargo aMTD Amino Acid 3′ Primer Protein ID Sequence 5′ Primer (5′→3′) (5′→3′) RF-01 165 ALAVPVALAIVP GGAATTC CATATG CCC AAG  CG GGATCC GTT TGA  OCT4 AAG AAG AGG AAG CTG GCG  ATG CAT GGG AGA GCC CTG GCG GTG CCG GTG GCG  CTG GCG ATT GTG CCG GCGGGACACCTGGCTTCGGATTTC 363 AVLAVAPALIVP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG GCG  GTG CTG GCG GTG GCG CCG  GCG CTG ATT GTG CCG GCGGGACACCTGGCTTCGGATTTC 405 LAAAVIPVAILP GGAATTC CATATG CCC AAG   AAG AAG AGG AAG CTG CTG  GCG GCG GCG GTG ATT CCG  GTG GCG ATT CTG CCG GCGGGACACCTGGCTTCGGATTTC 563 ALAVIVVPALAP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG GCG  CTG GCG GTG ATT GTG GTG  CCG GCG CTG GCG CCG GCGGGACACCTGGCTTCGGATTTC 889 ILVAAAPIAALP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG ATT  CTG GTG GCG GCG GCG CCG  ATT GCG GCG CTG CCG GCGGGACACCTGGCTTCGGATTTC 895 AIIIVVPAIAAP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG GCG  ATT ATT ATT GTG GTG CCG  GCG ATT GCG GCG CCG GCGGGACACCTGGCTTCGGATTTC 904 AVLAVVAPVVAP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG GCG  GTG CTG GCG GTG GTG GCG  CCG GTG GTG GCG CCG GCGGGACACCTGGCTTCGGATTTC

TABLE 39 Cargo aMTD Amino Acid 3′ Primer Protein ID Sequence 5′ Primer (5′→3′) (5′→3′) RF-02 161 AVIALPALIAAP GGAATTC CATATG CCC AAG  CG GGATCC CCT CGG  SOX2 AAG AAG AGG AAG CTG GCG  CTG CAC CGG CAC GGA GTG ATT GCG CTG CCG GCG  CTG ATT GCG GCG CCG GCAAATATTACCGTTTTCTAT 165 ALAVPVALAIVP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG GCG  CTG GCG GTG CCG GTG GCG  CTG GCG ATT GTG CCG GCAAATATTACCGTTTTCTAT 363 AVLAVAPALIVP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG GCG  GTG CTG GCG GTG GCG CCG  GCG CTG ATT GTG CCG GCAAATATTACCGTTTTGTAT 405 LAAAVIPVAILP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG CTG  GCG GCG GCG GTG ATT CCG  GTG GCG ATT CTG CCG GCAAATATTACCGTTTTCTAT 563 ALAVIVVPALAP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG GCG  CTG GCG GTG ATT GTG GTG  CCG GCG CTG GCG CCG GCAAATATTACCGTTTTCTAT 889 ILVAAAPIAALP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG ATT  CTG GTG GCG GCG GCG CCG  ATT GCG GCG CTG CCG GCAAATATTACCGTTTTCTAT 904 AVLAVVAPVVAP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG GCG  GTG CTG GCG GTG GTG GCG  CCG GTG GTG GCG CCG GCAAATATTACCGTTTTCTAT

TABLE 40 Cargo aMTD Amino Acid 3′ Primer Protein ID Sequence 5′ Primer (5′→3′) (5′→3′) RF-03 563 ALAVIVVPALAP GGAATTC CATATG CCC AAG  CG GGATCC CCT CGG  KLF4 AAG AAG AGG AAG CTG GCG  CTG CAC CGG CAC GGA CTG GCG GTG ATT GTG GTG  CCG GCG CTG GCG CCG GCAAATATTACCGTTTTCTAT

TABLE 41 Cargo aMTD Amino Acid 3′ Primer Protein ID Sequence 5′ Primer (5′→3′) (5′→3′) RF-04 161 AVIALPALIAAP GGAATTC CATATG CCC AAG  CG GGATCC CCT CGG  CMYC AAG AAG AGG AAG CTG GCG  CTG CAC CGG CAC GGA GTG ATT GCG CTG CCG GCG  CTG ATT GCG GCG CCG CCCCTCAACGTTAGCTTCACCAAC 165 ALAVPVALAIVP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG GCG  CTG GCG GTG CCG GTG GCG  CTG GCG ATT GTG CCG CCCCTCAACGTTAGCTTCACCAAC 363 AVLAVAPALIVP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG GCG  GTG CTG GCG GTG GCG CCG  GCG CTG ATT GTG CCG CCCCTCAACGTTAGCTTCACCAAC 405 LAAAVIPVAILP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG CTG  GCG GCG GCG GTG ATT CCG  GTG GCG ATT CTG CCG CCCCTCAACGTTAGCTTCACCAAC 563 ALAVIVVPALAP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG GCG  CTG GCG GTG ATT GTG GTG  CCG GCG CTG GCG CCG CCCCTCAACGTTAGCTTCACCAAC 889 ILVAAAPIAALP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG ATT  CTG GTG GCG GCG GCG CCG  ATT GCG GCG CTG CCG CCCCTCAACGTTAGCTTCACCAAC 895 AIIIVVPAIAAP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG GCG  ATT ATT ATT GTG GTG CCG  GCG ATT GCG GCG CCG CCCCTCAACGTTAGCTTCACCAAC 904 AVLAVVAPVVAP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG GCG  GTG CTG GCG GTG GTG GCG  CCG GTG GTG GCG CCG CCCCTCAACGTTAGCTTCACCAAC

TABLE 42 Cargo aMTD Amino Acid 3′ Primer Protein ID Sequence 5′ Primer (5′→3′) (5′→3′) RF-05 161 AVIALPALIAAP GGAATTC CATATG CCC AAG  ACGC GTCGAC CAC GTC  NANOG AAG AAG AGG AAG CTG GCG   TTC AGG TTG CAT GTT GTG ATT GCG CTG CCG GCG CTG ATT GCG GCG CCG AGTGTGGATCCAGCTTGTCCCCAA 405 LAAAVIPVAILP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG CTG  GCG GCG GCG GTG ATT CCG  GTG GCG ATT CTG CCG AGTGTGGATCCAGCTTGTCCCCAA 889 ILVAAAPIAALP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG ATT  CTG GTG GCG GCG GCG CCG  ATT GCG GCG CTG CCG AGTGTGGATCCAGCTTGTCCCCAA 895 AIIIVVPAIAAP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG GCG  ATT ATT ATT GTG GTG CCG  GCG ATT GCG GCG CCG AGTGTGGATCCAGCTTGTCCCCAA 904 AVLAVVAPVVAP GGAATTC CATATG CCC AAG  AAG AAG AGG AAG CTG GCG  GTG CTG GCG GTG GTG GCG  CCG GTG GTG GCG CCG AGTGTGGATCCAGCTTGTCCCCAA

TABLE 43 Cargo aMTD Amino Acid 3′ Primer Protein ID Sequence 5′ Primer (5′→3′) (5′→3′) RF-06 161 AVIALPALIAAP CCG GAATTC CCC AAG AAG ACGC GTCGAC ATT CTG  LIN28 AAG AGG AAG CTG GCG GTG  TGC CTC CGG GAG CAG ATT GCG CTG CCG GCG CTG  ATT GCG GCG CCG GGCTCCGTGTCCAACCAGCAGTTT 165 ALAVPVALAIVP CCG GAATTC CCC AAG AAG  AAG AGG AAG CTG GCG CTG  GCG GTG CCG GTG GCG CTG  GCG ATT GTG CCG GGCTCCGTGTCCAACCAGCAGTTT 563 ALAVIVVPALAP CCG GAATTC CCC AAG AAG  AAG AGG AAG CTG GCG CTG  GCG GTG ATT GTG GTG CCG  GCG CTG GCG CCG GGCTCCGTGTCCAACCAGCAGTTT 895 AIIIVVPAIAAP CCG GAATTC CCC AAG AAG  AAG AGG AAG CTG GCG ATT  ATT ATT GTG GTG CCG GCG  ATT GCG GCG CCG GGCTCCGTGTCCAACCAGCAGTTT

TABLE 44 Cargo aMTD Amino Acid 3′ Primer Protein ID Sequence 5′ Primer (5′→3′) (5′→3′) RF-07 563 ALAVIVVPALAP CCG GAATTC CCC AAG AAG ACGC GTCGAC GGA AGC  ZSCAN4 AAG AGG AAG CTG GCG CTG  TTC TGG TGT GGA GGG GCG GTG ATT GTG GTG CCG  GCG CTG GCG CCG GCTTTAGATCTAAGAACCATATTT

<6-2> Expression and Purification of Histidine-Tagged RF Recombinant Proteins

The transformant was cultured in LB medium containing 50 ug/ml of kanamycin, and the transformant was inoculated in 7 ml of LB medium at 37° C. overnight. The incubated transformant was inoculated in 700 ml of LB medium at 37° C. until OD₆₀₀ reached 0.5. The medium was added with 0.7 mM isopropyl-β-D-thiogalactoside (IPTG) as a protein expression inducer, and further incubated at 37° C. for 3 hours. The medium was centrifuged at 4° C. and 8,000×g for 10 minutes, and a supernatant was discarded to recover a cell pellet. The pellet was loaded on SDS-PAGE to analyze expression levels. The pellet was re-suspended in the lysis buffer (50 mM NaH₂PO₄, 10 mM Imidazol, 300 mM NaCl, pH 8.0). This suspension was disrupted with sonication to the cells. The disrupted cells were centrifuged at 4° C. and 15,000×g for 30 minutes to obtain a soluble fraction and an insoluble fraction. Recombinant proteins are supposed to be purified by Ni²+ affinity chromatography as directed by the supplier (Qiagen, Germany) in the natural condition. After purification, they will be changed to a Dulbecco's Modified Eagle's Medium (DMEM), (Hyclone, USA).

Example 7. Determination of Solubility/Yield of RF Recombinant Proteins

The aMTD-fused RF proteins (OCT4, SOX2, CMYC, KLF4, NANOG, LIN28 and ZSCAN4) containing SDs (SA and SB) were individually cloned, expressed, purified and prepared in a soluble form. The solubility and yield of each recombinant protein fused to the aMTD with the SD were determined.

Each RF recombinant protein was determined for their size (number of amino acids), yield (mg/L) and solubility on 10% SDS-PAGE gel and stained with Coomassie Brilliant Blue.

Consequently, the SD was confirmed to influence improvement of solubility and yield of the RF recombinant proteins.

<7-1> OCT4 Recombinant Proteins

Each recombinant protein; HO, HNM₅₆₃0 and HNM₅₆₃OSB (FIG. 18) was determined for their size (number of amino acids), yield (mg/L) and solubility.

As shown in FIG. 19 (top), the proteins were observed as a single band.

As shown in FIG. 19 (bottom), it was confirmed that HNM₅₆₃OSB showed improved yield and solubility, compared to HO and HNM₅₆₃0, and HNM₅₆₃OSB was determined as a basic structure of the OCT4 recombinant protein.

<7-2> SOX2 Recombinant Proteins

Each recombinant protein; HS, HNM₅₆₃S, HNM₅₆₃SSB, HNM₅₆₃SASSBSB and HNM₅₆₃SASSASBSASB (FIG. 20) was determined for their size (number of amino acids), yield (mg/L) and solubility.

As shown in FIG. 21 (top), the proteins were observed as a single band.

As shown in FIG. 21 (bottom), it was confirmed that HNM₅₆₃SSB, HNM₅₆₃SASSBSB and HNM₅₆₃SASSASBSASB showed improved yield and solubility, compared to HS and HNM₅₆₃S, and HNM₅₆₃SASSASBSASB was determined as a basic structure of the SOX2 recombinant protein.

<7-3> KLF4 Recombinant Proteins

Each recombinant protein; HK, HNM₅₆₃K, HNM₅₆₃KSB, HNM₅₆₃SAKSBSB and HNM₅₆₃SAKSASBSASB (FIG. 22) was determined for their size (number of amino acids), yield (mg/L) and solubility.

As shown in FIG. 23 (top), the proteins were observed as a single band.

As shown in FIG. 23 (bottom), it was confirmed that HNM₅₆₃KSB, HNM₅₆₃SAKSBSB and HNM₅₆₃SAKSASBSASB showed improved yield and solubility, compared to HK and HNM₅₆₃K, and HNM₅₆₃SAKSASBSASB was determined as a basic structure of the KLF4 recombinant protein.

<7-4> CMYC Recombinant Proteins

Each recombinant protein; HM, HNM₅₆₃M and HNM₅₆₃MSB (FIG. 24) was determined for their size (number of amino acids), yield (mg/L) and solubility.

As shown in FIG. 25 (top), the proteins were observed as a single band.

As shown in FIG. 25 (bottom), it was confirmed that HNM₅₆₃MSB showed improved yield and solubility, compared to HM and HNM₅₆₃M, and HNM₅₆₃MSB was determined as a basic structure of the CMYC recombinant protein.

<7-5> NANOG Recombinant Proteins

Each recombinant protein; HN, HNM₁₆₁N and HNM₁₆₁NSB (FIG. 26) was determined for their size (number of amino acids), yield (mg/L) and solubility.

As shown in FIG. 27 (top), the proteins were observed as a single band.

As shown in FIG. 27 (bottom), it was confirmed that HNM₁₆₁NSB showed improved yield and solubility, compared to HN and HNM₁₆₁N, and HNM₁₆₁NSB was determined as a basic structure of the NANOG recombinant protein.

<7-6> LIN28 Recombinant Proteins

Each recombinant protein; HL, HNM₅₆₃L and HNM₅₆₃LSB (FIG. 28) was determined for their size (number of amino acids), yield (mg/L) and solubility.

As shown in FIG. 29 (top), the proteins were observed as a single band.

As shown in FIG. 29 (bottom), it was confirmed that HNM₅₆₃LSB showed improved yield and solubility, compared to HL and HNM₅₆₃L, and HNM₅₆₃LSB was determined as a basic structure of the LIN28 recombinant protein.

<7-7> ZSCAN4 Recombinant Proteins

Each recombinant protein; HZ, HNM₅₆₃Z and HNM₅₆₃ZSB (FIG. 30) was determined for their size (number of amino acids), yield (mg/L) and solubility.

As shown in FIG. 31 (top), the proteins were observed as a single band.

As shown in FIG. 31 (bottom), it was confirmed that HNM₅₆₃ZSB showed improved yield and solubility, compared to HZ and HNM₅₆₃Z, and HNM₅₆₃ZSB was determined as a basic structure of the ZSCAN4 recombinant protein.

Example 8. Determination of Optimal aMTD for iCP-RF Recombinant Proteins

To increase the cell-permeability of the RF recombinant proteins, aMTD fused to RF protein was replaced various aMTDs. The yield and solubility of each RF recombinant protein fused various aMTDs were measured. Consequently, it was confirmed that both aMTD and SD improved solubility and yield of the RF proteins, and optimal aMTD for each RF recombinant protein was determined.

<8-1> SOX2 Recombinant Proteins

In the same manner as in Example 6, aMTD₁₆₁, aMTD₁₆₅, aMTD₃₆₃, aMTD₄05, aMTD₅₆₃, aMTD₈₈₉, and aMTD₉₀₄-fused SOX2 recombinant proteins were prepared (FIG. 32). Yield and solubility of the SOX2 recombinant proteins were measured in the same manner as in Example 7. Primers used are as given in Table 39.

As shown in FIG. 33 (top), the proteins were observed as a single band.

As shown in FIG. 33 (bottom), all the SOX2 recombinant proteins fused with aMTDs showed high solubility. The aMTD₅₆₃-fused SOX2 recombinant protein was found to have the highest yield and solubility. Consequently, the aMTD₅₆₃-fused SOX2 recombinant protein was determined as iCP-SOX2 recombinant protein.

<8-2> NANOG Recombinant Proteins

In the same manner as in Example 6, aMTD₁₆₁, aMTD₄₀₅, aMTD₈₈₉, aMTD₈₉₅, and aMTD₉₀₄-fused NANOG recombinant proteins were prepared (FIG. 34). Yield and solubility of the NANOG recombinant proteins were measured in the same manner as in Example 7. Primers used are as given in Table 42.

As shown in FIG. 35 (top), the proteins were observed as a single band.

As shown in FIG. 35 (bottom), all the NANOG recombinant proteins fused with aMTDs showed high solubility. The aMTD₁₆₁-fused NANOG recombinant protein was found to have the highest yield and solubility. Consequently, the aMTD₁₆₁-fused NANOG recombinant protein was determined as iCP-NANOG recombinant protein.

<8-3> OCT4 Recombinant Proteins

In the same manner as in Example 6, aMTD₁₆₅, aMTD₃₆₃, aMTD₄₀₅, aMTD₅₆₃, aMTD₈₈₉, aMTD₈₉₅, and aMTD₉04-fused OCT4 recombinant proteins were prepared (FIG. 36). Yield and solubility of the OCT4 recombinant proteins were measured in the same manner as in Example 7. Primers used are as given in Table 38.

All the OCT4 recombinant proteins fused with aMTDs showed high solubility. The aMTD₅₆₃-fused OCT4 recombinant protein was found to have the highest yield and solubility. Consequently, the aMTD₅₆₃-fused OCT4 recombinant protein was determined as iCP-OCT4 recombinant protein.

<8-4> CMYC Recombinant Proteins

In the same manner as in Example 6, aMTD₁₆₁, aMTD₁₆₅, aMTD₃₆₃, aMTD₄₀₅, aMTD₅₆₃, aMTD₈₈₉, aMTD₈₉₅, and aMTD₉₀₄-fused CMYC recombinant proteins were prepared (FIG. 37). Yield and solubility of the CMYC recombinant proteins were measured in the same manner as in Example 7. Primers used are as given in Table 41.

All the CMYC recombinant proteins fused with aMTDs showed high solubility. The aMTD₅₆₃-fused CMYC recombinant protein was found to have the highest yield and solubility. Consequently, the aMTD₅₆₃-fused CMYC recombinant protein was determined as iCP-CMYC recombinant protein.

<8-5> LIN28 Recombinant Proteins

In the same manner as in Example 6, aMTD₁₆₁, aMTD₁₆₅, aMTD₅₆₃, and aMTD₅₉₅-fused LIN28 recombinant proteins were prepared (FIG. 38). Yield and solubility of the LIN28 recombinant proteins were measured in the same manner as in Example 7. Primers used are as given in Table 43.

All the LIN28 recombinant proteins fused with aMTDs showed high solubility. The aMTD₅₆₃-fused LIN28 recombinant protein was found to have the highest yield and solubility. Consequently, the aMTD₅₆₃-fused LIN28 recombinant protein was determined as iCP-LIN28 recombinant protein.

<8-6> KLF4 Recombinant Proteins and ZSCAN4 Recombinant Proteins

aMTD₅₆₃-fused KLF4 recombinant protein was determined as iCP-KLF4 recombinant protein, and aMTD₅₆₃-fused ZSCAN4 recombinant protein was determined as iCP-ZSCAN4 recombinant protein.

9. Determination of Cell-Permeability of iCP-RF Recombinant Proteins

Cell-permeability and intranuclear delivery of the iCP-RF recombinant proteins; iCP-OCT4, iCP-SOX2, iCP-CMYC, iCP-KLF4 and iCP-LIN28 were examined by flow cytometry and confocal laser microscopy. Overall, it was confirmed that the aMTD-fused RF proteins had improved cell-permeability and they were efficiently delivered into the nuclei of the cells.

<9-1> Flow Cytometry

For cell permeability, the iCP-RF recombinant proteins were conjugated to FITC according to the manufacturer's instructions (Pierce Chemical, Rockford, Ill.). RAW 264.7 cells (ATCC, USA) were treated with 10 uM FITC-labeled RF proteins for 1 hour at 37° C., washed three times with cold PBS, treated with proteinase K (10 ug/ml) for 20 min at 37° C. to remove cell-surface bound proteins and subjected to fluorescence-activated cell sorting (FACS) analysis (FACSCalibur; BD, Franklin Lakes, N.J.).

As shown in FIG. 39, aMTD-fused OCT4/SOX2/KLF4/LIN28/CMYC recombinant proteins (aMTD-RF-SD) showed improved cell-permeability, compared to the RF recombinant protein without aMTD (RF). Consequently, it was confirmed that the iCP-RF recombinant proteins are provided with excellent cell permeability by aMTD.

<9-2> Confocal Laser Microscope

NIH3T3 cells were seeded in 8-well chamber, 2×10⁴ cells/well. After day, the NIH3T3 cells were treated with 10 uM FITC-labeled iCP-RF recombinant proteins for 2 hours, and then fixed in 2% paraformaldehyde for 10 minutes. Then, 1 or 2 drops of a DAPI-containing mounting solution (Vector Laboratories, Inc., VECTASHIELD® MOUNTING MEDIUM with DAPI, Catalog Number H-1200), the cells were observed under a confocal laser scanning microscope.

As shown in FIG. 40, it was found that the iCP-RF recombinant proteins showed cell-permeability and intranuclear delivery. These results suggest that the iCP-RF recombinant proteins have excellent cell permeability and induce delivery of RF proteins into the nucleus to show the biological activity (generation of iPSCs).

Example 10. Determination of Biological Activity of iCP-RF Recombinant Proteins in Reporter Cells

To measure the biological activity of the RF recombinant proteins in the nucleus, the constructed luciferase vector regulating luciferase expression was used (FIG. 41a ).

Human HeLa cells were transfected with 300 ng of the luciferase expression vector. After 24 hours, the cells were treated with 0.1, 0.5, 1, and 2 uM of each iCP-RF recombinant protein for 6 hours. Each of the cells treated with the iCP-RF recombinant proteins was lysed using a 1× passive lysis buffer (Promega) and incubated at room temperature for 15 minutes. Luciferase activity was measured using a Dual-luciferase reporter assay (Promega) and a LUMIstar omega luminometer (BMG LABTECH) according to the manufacturer's instructions.

<10-1> iCP-OCT4 Recombinant Proteins

As shown in FIG. 41b , it was confirmed that the iCP-OCT4 recombinant protein bound with luciferase promoter in the nucleus, and expressed luciferase.

Further, when 0.5 uM of the iCP-OCT4 recombinant protein was treated, the activity was 38-fold higher than that of the control (only vector).

<10-2> iCP-SOX2 Recombinant Proteins

As shown in FIG. 42, it was confirmed that the iCP-SOX2 recombinant protein bound with luciferase promoter in the nucleus, and expressed luciferase.

Further, when 0.1 uM of the iCP-SOX2 recombinant protein was treated, the activity was 27-fold higher than that of the control (only vector).

<10-3> iCP-KLF4 Recombinant Proteins

As shown in FIG. 43, it was confirmed that the iCP-KLF4 recombinant protein bound with luciferase promoter in the nucleus, and expressed luciferase.

Further, when 0.1 uM of the iCP-KLF4 recombinant protein was treated, the activity was 22-fold higher than that of the control (only vector).

<10-4> iCP-CMYC Recombinant Proteins

As shown in FIG. 44, it was confirmed that the iCP-CMYC recombinant protein bound with luciferase promoter in the nucleus, and expressed luciferase.

Further, when 0.5 uM of the iCP-CMYC recombinant protein was treated, the activity was 34-fold higher than that of the control (only vector).

<10-5> iCP-NANOG Recombinant Proteins

As shown in FIG. 45, it was confirmed that the iCP-NANOG recombinant protein bound with luciferase promoter in the nucleus, and expressed luciferase.

Further, when 0.5 uM of the iCP-NANOG recombinant protein was treated, the activity was 27-fold higher than that of the control (only vector).

<10-6> iCP-LIN28 Recombinant Proteins

As shown in FIG. 46, it was confirmed that the iCP-LIN28 recombinant protein bound with luciferase promoter in the nucleus, and expressed luciferase.

Further, when 0.1 uM of the iCP-LIN28 recombinant protein was treated, the activity was 30-fold higher than that of the control (only vector).

Example 11. Protocol for iPSC-Like Colony Formation by iCP-RF Recombinant Proteins

To generate iPSCs with high efficiency, treatment conditions of the iCP-RF recombinant proteins (iCP-OCT4, iCP-SOX2, iCP-KLF4, iCP-CMYC, iCP-LIN28, iCP-NANOG and iCP-ZSCAN4) were controlled to carry out Protocol 1 to Protocol 5.

<11-1> Protocol 1

Human umbilical vein endothelial cells (HUVEC) were treated with each 0.1 uM of the RF recombinant proteins (iCP-OCT4, iCP-SOX2, iCP-KLF4, iCP-CMYC and iCP-LIN28) for 6 hours a day for total 5 days. After 3 days, the cells began to form colonies (FIG. 47, top). To examine whether the colonies were iPSC-like colonies, alkaline phosphatase (AP) staining (Life Technologies) was performed according to the manufacturer's instructions.

As shown in FIG. 47 (bottom), the colonies formed at 3 days exhibited AP positive fluorescence, indicating iPSC-like colonies. As a result, when somatic cells were treated with 0.1 uM of the RF recombinant proteins (iCP-OCT4, iCP-SOX2, iCP-KLF4, iCP-CMYC and iCP-LIN28), iPSCs generation was observed at 3 days.

<11-2> Protocol 2

Human umbilical vein endothelial cells (HUVEC) were treated with each 0.5 uM of the RF recombinant proteins (iCP-OCT4, iCP-SOX2, iCP-KLF4, iCP-CMYC and iCP-LIN28) for 6 hours a day for total 5 days. After 3 days, the cells began to form colonies (FIG. 48, top). To examine whether the colonies were iPSC-like colonies, alkaline phosphatase (AP) staining (Life Technologies) was performed according to the manufacturer's instructions.

As shown in FIG. 48 (bottom), the colonies formed at 3 days exhibited AP positive fluorescence, indicating iPSC-like colonies. As a result, when somatic cells were treated with 0.5 uM of the RF recombinant proteins (iCP-OCT4, iCP-SOX2, iCP-KLF4, iCP-CMYC and iCP-LIN28), iPSCs generation was observed at 3 days.

<11-3> Protocol 3

BJ cells (Human fibroblast) were treated with each 0.5 uM of the RF recombinant proteins (iCP-OCT4, iCP-SOX2, iCP-KLF4, iCP-CMYC, iCP-LIN28 and iCP-ZSCAN4) for 6 hours a day for total 10 days. After 3 days, the cells began to form colonies (FIG. 49, top). In order to examine whether the colonies were iPSC-like colonies, alkaline phosphatase (AP) staining (Life Technologies) was performed according to the manufacturer's instructions.

As shown in FIG. 49 (bottom), the colonies formed at 3 days exhibited AP positive fluorescence, indicating iPSC-like colonies, and the colonies maintained for 7 days. As a result, when somatic cells were treated with 0.5 uM of the RF recombinant proteins (iCP-OCT4, iCP-SOX2, iCP-KLF4, iCP-CMYC, iCP-LIN28 and iCP-ZSCAN4), iPSCs generation was observed at 3 days.

<11-4> Protocol 4

BJ cells (Human fibroblast) were treated with each 0.5 uM of the RF recombinant proteins (iCP-OCT4, iCP-CMYC and iCP-NANOG) for 6 hours a day for total 10 days. After 6 days, the cells began to form colonies (FIG. 50, top). To examine whether the colonies were iPSC-like colonies, alkaline phosphatase (AP) staining (Life Technologies) was performed according to the manufacturer's instructions.

As shown in FIG. 50 (bottom), the colonies formed at 6 days exhibited AP positive fluorescence, indicating iPSC-like colonies. As a result, when somatic cells were treated with 0.5 uM of the RF recombinant proteins (iCP-OCT4, iCP-CMYC and iCP-NANOG), iPSCs generation was observed at 6 days.

<11-5> Protocol 5

4 groups of Detroit 573 cells (Human fibroblast) were treated with each 0.00025, 0.0005, 0.00125 or 0.0025 uM of the RF recombinant proteins (iCP-OCT4, iCP-SOX2, iCP-KLF4, iCP-CMYC, iCP-NANOG, iCP-LIN28 and iCP-ZSCAN4) for 6 hours a day for total 7 days. After 7 days, the cells began to form colonies (FIG. 51, top). To examine whether the colonies were iPSC-like colonies, alkaline phosphatase (AP) staining (Life Technologies) and immunocytochemistry were performed according to the manufacturer's instructions.

As shown in FIG. 51 (bottom), the colonies formed at 7 days were observed under a microscope. As a result, when all 4 groups of somatic cells were treated with each 0.00025, 0.0005, 0.00125 or 0.0025 uM of the RF recombinant proteins (iCP-OCT4, iCP-SOX2, iCP-KLF4, iCP-CMYC, iCP-NANOG, iCP-LIN28 and iCP-ZSCAN4), iPSCs generation was observed at 7 days.

In conclusion, terminally differentiated somatic cells can be reprogrammed and dedifferentiated to iPSC by the combination of the RF recombinant proteins (iCP-OCT4, iCP-SOX2, iCP-CMYC, iCP-KLF4, iCP-NANOG, iCP-LIN28 and iCP-ZSCAN4).

Example 12. Determination of Activity of iPSC-Like Colony Formed by iCP-RF Recombinant Proteins

To determinate the activity of dedifferentiated iPSCs, OCT4 and TRA-1-81 expressed in iPSCs were examined.

The iPSC-like colonies formed in the same manner as in Example <11-4> were expanded and the iPSC-like colonies were maintained for 30 days (FIG. 52, top). The colonies were transferred to 8-well chamber slide (NUNC, Waltham, Mass.) using a capillary glass tube. After day, the chamber slide was washed with PBS twice. The colonies were fixed in 2% paraformaldehyde for 20 minutes, and washed with PBS twice. The colonies treated with 0.1% Triton X-100 for 5 minutes, and washed with PBS twice. The colonies incubated with 2% BSA at room temperature for 1 hr, and incubated with a goat polyclonal anti-OCT4 or anti-Tra-1-81 antibody (1:1000 dilution in 2% BSA/PBS) at 4° C. o/n. The colonies were washed with PBS twice and incubated with an Alexa Fluor 488 rabbit anti-goat IgG secondary antibody (1:1000 dilution in 2% BSA/PBS) at room temperature for 1 hr. The nucleus were stained with 300 nM DAPI (4, 6-diamidino-2-phenylindele) in the dark at room temperature for 5 minutes, and then washed with PBS three times. The cells were treated with a mounting medium (Vector Laboratories, Inc., VECTASHIELD® MOUNTING MEDIUM with DAPI, Catalog Number H-1200) and covered with a coverslip. After 15 minutes, the cells were observed under a confocal microscope.

As shown in FIG. 52 (bottom), overall colonies showed OCT4 and TRA-1-81 expressions. As a result, it was confirmed that iPSCs were generated by treatment of somatic cells with the iCP-RF recombinant proteins, and the iPSCs were maintained for 30 days. These results suggest that the RF recombinant proteins provide the reprogramming activity for dedifferentiation of somatic cells.

Example 13. Statistical Analysis

All experimental data using cultured cells are expressed as means S.D. for at least three independent experiments. Statistical significance is evaluated using a two-tailed Student's t-test or ANOVA method. Experimental differences between groups are assessed using paired Student's t-tests. For animal experiments, ANOVA is used for comparing between and within groups to determine the significance. Differences with p<0.05 are considered to be statistically significant.

Those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in different forms without departing from the essential characteristics thereof. Therefore, it should be understood that the disclosed embodiments are not limitative, but illustrative in all aspects. The scope of the present invention is made to the appended claims rather than to the foregoing description, and all variations which come within the range of equivalency of the claims are therefore intended to be embraced therein. 

The invention claimed is:
 1. A recombinant protein, which comprises a RF protein selected from the group consisting of OCT4, SOX2, CMYC, KLF4, NANOG, LIN28 and ZSCAN4, and an advanced macromolecule transduction domain (aMTD) being composed of 12 amino acid residues and having improved cell or tissue permeability, wherein the aMTD has an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 43, 84, 96, 131, 223, 226, and 233, or the aMTD is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 279, 283, 324, 336 371, 463, 466, and
 473. 2. The recombinant protein according to claim 1, wherein one or more solubilization domain (SD)(s) are further fused to the end(s) of one or more of the RF protein and the aMTD, wherein the one or more SD(s) independently have an amino acid sequence selected from the group consisting of SEQ ID NOs: 798-804.
 3. The recombinant protein according to claim 2, wherein the recombinant protein is represented by any one of the following structural formula: A-B—C and A-C—B—C wherein A is an advanced macromolecule transduction domain (aMTD) having improved cell or tissue permeability, B is a RF protein selected from the group consisting of OCT4, SOX2, CMYC, KLF4, NANOG, LIN28 and ZSCAN4, and C is a solubilization domain (SD); and wherein one or more SD(s) independently have an amino acid sequence selected from the group consisting of SEQ ID NOs: 798-804.
 4. The recombinant protein according to claim 1, wherein the RF protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 816 to
 822. 5. The recombinant protein according to claim 4, wherein the RF protein is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 823 to
 829. 6. The recombinant protein of claim 2, wherein the SD(s), independently, are encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 805 to
 811. 7. The recombinant protein according to claim 1, wherein the fusion is formed via a peptide bond or a chemical bond.
 8. The recombinant protein according to claim 1, wherein the recombinant protein is used for the generation of induced pluripotent stem cells (iPSCs) from somatic cells.
 9. A preparing method of the recombinant protein of claim 1 comprising: culturing a transformant comprising an exogenous polynucleotide encoding the recombinant protein of claim 1 in a culture medium to produce the recombinant protein; and recovering the recombinant protein expressed by the culturing.
 10. A method of inducing generation of iPSCs from somatic cells comprising: treating the somatic cells with an effective amount of the recombinant protein according to claim
 1. 11. The recombinant protein of claim 3, wherein the SD(s), independently, are encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 805 to
 811. 12. A preparing method of the recombinant protein comprising: culturing a transformant comprising a n exogenous polynucleotide encoding the recombinant protein of claim 3 in a culture medium to produce the recombinant protein; and recovering the recombinant protein expressed by the culturing.
 13. The method according to claim 9, wherein the polynucleotide sequence is selected from the group consisting of SEQ ID NOs: 831, 837, 843, 849, 855, 861, and
 867. 14. The method according to claim 12, wherein the polynucleotide sequence is selected from the group consisting of SEQ ID NOs: 833, 839, 845, 851, 857, 863, and
 869. 